Revision 2410 Sep 2013 - Main.EdEng

Contents

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids
Data re-analysis
- combine untilted stacks from 4h and 16 h post gelfiltration IR in DDM taken in April-May 2012
  - 16hr stack : 20120417IRa8_16h.spd and contains 5234 particles
  - 4 hr stack : 20120510IRg1_04h.spd and contains 2872 patticles
  - combine.spd : combined stack wtih 8106 images

June 2013

Ed,

Here are the .box files for the IR EM images. I went picked particles from a little over 100 images. I had to rename some files so I included a key in the folder but I will explain it here too. Essentially, I have three folders from images. Folders "P3 Round 1" and "P3 Round 2" from 6/12/13 and "P3" from 6/19/13. The files were renamed to the following... P3_A_001 to P3_A_005 = Images from 6/12/13 P3 Round 1 P3_B_001 to P3_B_050 = Images from 6/12/13 P3 Round 2 P3_C_001 to P3_C_150 = Images from 6/19/13

I am attempting to continue with analysis but I have been receiving a error stating "The data area passed to a system call is too small". I'm not completely sure what that error means but my guess is my laptop might not have the processing power necessary to complete the function. I might have to install EMAN2 on our lab's desktop computer and try it on that. I'll keep you updated on my progress.

Richie

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

* zip.zip: zip.zip

META FILEATTACHMENT	attachment="zip.zip" attr="" comment="" date="1366301468" name="zip.zip" path="zip.zip" size="23936151" stream="zip.zip" user="Main.EdEng" version="1"

Added:

>
>

META TOPICMOVED	by="EdEng" date="1378827315" from="Main.CemProjectsEng" to="Main.CemEngProjects"

Revision 2324 Jun 2013 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids
Data re-analysis
- combine untilted stacks from 4h and 16 h post gelfiltration IR in DDM taken in April-May 2012
  - 16hr stack : 20120417IRa8_16h.spd and contains 5234 particles
  - 4 hr stack : 20120510IRg1_04h.spd and contains 2872 patticles
  - combine.spd : combined stack wtih 8106 images

Added:

>
>

June 2013

Ed,

Here are the .box files for the IR EM images. I went picked particles from a little over 100 images. I had to rename some files so I included a key in the folder but I will explain it here too. Essentially, I have three folders from images. Folders "P3 Round 1" and "P3 Round 2" from 6/12/13 and "P3" from 6/19/13. The files were renamed to the following... P3_A_001 to P3_A_005 = Images from 6/12/13 P3 Round 1 P3_B_001 to P3_B_050 = Images from 6/12/13 P3 Round 2 P3_C_001 to P3_C_150 = Images from 6/19/13

I am attempting to continue with analysis but I have been receiving a error stating "The data area passed to a system call is too small". I'm not completely sure what that error means but my guess is my laptop might not have the processing power necessary to complete the function. I might have to install EMAN2 on our lab's desktop computer and try it on that. I'll keep you updated on my progress.

Richie

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

* zip.zip: zip.zip

META FILEATTACHMENT	attachment="zip.zip" attr="" comment="" date="1366301468" name="zip.zip" path="zip.zip" size="23936151" stream="zip.zip" user="Main.EdEng" version="1"

Revision 2218 Apr 2013 - Main.EdEng

Contents

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids
Data re-analysis
- combine untilted stacks from 4h and 16 h post gelfiltration IR in DDM taken in April-May 2012
  - 16hr stack : 20120417IRa8_16h.spd and contains 5234 particles
  - 4 hr stack : 20120510IRg1_04h.spd and contains 2872 patticles
  - combine.spd : combined stack wtih 8106 images

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Added:

>
>

* zip.zip: zip.zip

META FILEATTACHMENT	attachment="zip.zip" attr="" comment="" date="1366301468" name="zip.zip" path="zip.zip" size="23936151" stream="zip.zip" user="Main.EdEng" version="1"

Revision 2108 Nov 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids
Data re-analysis
- combine untilted stacks from 4h and 16 h post gelfiltration IR in DDM taken in April-May 2012
  - 16hr stack : 20120417IRa8_16h.spd and contains 5234 particles

Changed:

<
<

- - 4 hr stack : 20120417IRa8_04h.spd and contains 2872 patticles

>
>

- - 4 hr stack : 20120510IRg1_04h.spd and contains 2872 patticles

Added:

>
>

- - combine.spd : combined stack wtih 8106 images

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Revision 2008 Nov 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids

Changed:

<
<

>
>

Data re-analysis
- combine untilted stacks from 4h and 16 h post gelfiltration IR in DDM taken in April-May 2012

Added:

>
>

- - 16hr stack : 20120417IRa8_16h.spd and contains 5234 particles
  - 4 hr stack : 20120417IRa8_04h.spd and contains 2872 patticles

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Revision 1908 Nov 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - February 23, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

February 23, 2012

First email from Steve

Changed:

<
<

March 9, 2012

>
>

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

Changed:

<
<

April 6, 2012

>
>

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

Changed:

<
<

May 4, 2012

>
>

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

Changed:

<
<

June 12, 2012

>
>

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Revision 1808 Nov 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Changed:

<
<

Initial meeting

>
>

Initial meeting

Steve Hubbard/Todd Miller

Added:

>
>

February 23, 2012

First email from Steve

March 9, 2012

JEOL2100 with Richie
First EM session with Richie
- 20120309_j2100-IR

April 6, 2012

* F20 with Richie

- 20120406_F20-IR
- 20120416_F20-IR
- 20120417_F20-IR
- 20120419_F20-IR

May 4, 2012

F20 with Richie
- 20120510_F20-IR
- 20120521-2_F20_IR
- 20120525IRrct

June 12, 2012

JEOL2100 with Richie
- 20120611_j2100-IR
- 20120613_F20_IR

June 20, 2012

F20 with Richie

July 10, 2012

Changed:

<
<

Trip to Miller lab @ StonyBrook^?

>
>

Trip to Miller lab @ StonyBrook^?

August 21, 2012

Added:

>
>

JEOL2100 with Richie

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

Added:

>
>

September 26, 2012

JEOL2100 with Richie

November 07-08, 2012

Making grids

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Revision 1722 Oct 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Deleted:

<
<

META FILEATTACHMENT	attachment="rs_tmv_awash_004.zip" attr="" comment="" date="1350926919" name="rs_tmv_awash_004.zip" path="rs_tmv_awash_004.zip" size="19419493" stream="rs_tmv_awash_004.zip" user="Main.RinkuJain" version="1"

Revision 1622 Oct 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Added:

>
>

META FILEATTACHMENT	attachment="rs_tmv_awash_004.zip" attr="" comment="" date="1350926919" name="rs_tmv_awash_004.zip" path="rs_tmv_awash_004.zip" size="19419493" stream="rs_tmv_awash_004.zip" user="Main.RinkuJain" version="1"

Revision 1501 Oct 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC
  - Dirk Remus
  - Prasad Jallepalli
    - July 10, 2012
    - July 23 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 - 600 kDa
Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample
1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Added:

>
>

RCT

# rct macro v2
# 05-21-12
# should have opened file for writing
# sould be eucentric on square
# should have set defocu target
# should be in low dose
# roll buffer limit set to M or less
# this one is for plain carbon -- no alignment to hole is done
# no pause if drift fails, continues macro 

MyAngle = 50  # set as desired
interval = 5 # set interval between photos for drift measurement
times = 8 # number of pictures
crit = 0.5  # drift must fall below this (nm/sec)
shot = Trial # type of image to take for drift measurement

T
copy A O # save current position
WalkUpTo $MyAngle

T   # to minimize image shift   
ResetImageShift
T
AlignTo B

Delay $interval
G
G  # autofocus twice

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

WalkUpto 0
ResetImageShift
G
G
AlignTo O

$shot
loop $times index
   $shot
   AlignTo B
   ReportAlignShift
   ClearAlignment
   dx = $reportedValue3
   dy = $reportedValue4
   dist = sqrt  $dx * $dx + $dy * $dy
   echo Distance = $dist nm
   if $dist < $crit
      echo drift is low enough after shot $index
      break
   endif
   if $index < $times
      echo warning drift is high in this image
   endif
EndLoop
R
S

#

Set ALLOWTOPICVIEW =

Revision 1422 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC
  - Dirk Remus

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Changed:

<
<

Mcm2-7

>
>

Mcm2-7 - 600 kDa

Added:

>
>

Notes:
Purification: Endogenous protein purified from yeast. Enriched with DNA coupled to magnetic beads. ORC (?300 kDa complex) and Cdc6 (60KDa protein) required to lead protein onto DNA.

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

Added:

>
>

grids: nitrocellulose/carbon

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain

Changed:

<
<

3A

15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain

No carbon

1A2

40

10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain

Smaller particles, heterogeneous sample, Double hexamer falling apart?

>
>

3A	0	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	No carbon
1A2	40	10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain	Smaller particles, aggregates, overall a heterogeneous sample, Double hexamer falling apart?
1AgridI	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	larger particles, aggregates, overall a heterogeneous sample

Added:

>
>

1AgridII	20	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	somewhere in between 1A2 and 1AgridI
1AgridIII	15	15 sec glow discharge, 2 uL sample, 4 min incubation, 1 wash, 30 sec stain	aggregation and no apparent single particles

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 1322 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC
  - Dirk Remus

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7

Added:

>
>

buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain

Changed:

<
<

3A

15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain

>
>

3A

15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain

No carbon

Added:

>
>

1A2

40

10 sec glow discharge, 2 uL sample, 5 min incubation, 2 washes, 30 sec stain

Smaller particles, heterogeneous sample, Double hexamer falling apart?

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 1222 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012
- MSKCC
  - Dirk Remus
    - August 9, 2012
    - August 15, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Added:

>
>

August 22, 2012

2nd 120kV session with JulienGros

Mcm2-7 buffer:

1A	100 mM KGlutamate, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide
3A	100 mM NaCl^?, 25 mM HEPES, pH 7.6, 10 mM MgAcetate^?, 5% glycerol, 1mM ATP, 2mM BME, 250 ng/uL FLAG peptide

Grid	Images	Preparation conditions	Comments
1A	10	15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain	Almost no carbon, the 2 grid squares with carbon had precipitate and too much stain
3A		15 sec glow discharge, 3 uL sample, 5 min incubation, 30 sec stain

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 1122 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

Changed:

<
<

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact O4 - PTA 2% in purification buffer -

>
>

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact
O4 - PTA 2% in purification buffer - tmv tracer - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 1021 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers?

Added:

>
>

K6 - 30 sec stain, 3 washes - some areas of pulled stain with good contrast

K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

Added:

>
>

O2 - PTA 2% 30 sec stain, 1 min incubation - tmv tracer - different morphology, more linear and compact O4 - PTA 2% in purification buffer -

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 921 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
    - August 21, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

Changed:

<
<

K2 - 30 sec stain - too light, particles and perhaps some monomers? K10 - 60 sec stain

>
>

K2 - 30 sec stain - too light, particles and perhaps some monomers?
K10 - 60 sec stain - better, but dark areas too much stain and light areas too light

Added:

>
>

L1 - 45 sec stain - light blotting, 1 min incubation - better concentration of particles but not stained evenly
L3 - 30 sec stain - no blotting, 2 min incubation - not stained well, aggregates of protein seen

M4 - carbon sandwhich, 2 min incubation, 30sec stain - undried stain

N1 - tmv tracer - 1 min incubation, unblotted - too much stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 821 Aug 2012 - Main.RinkuJain

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

Added:

>
>

August 21, 2012

IR from transient transfections - 90 million cells vs 1.5 billion

K2 - 30 sec stain - too light, particles and perhaps some monomers? K10 - 60 sec stain

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2
3 - sample 3

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 716 Aug 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
- MSKCC
  - Dirk Remus
    - August 9, 2012
    - August 15, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Mcm2-7 - double hexamers around DNA >600kDa

Added:

>
>

three different buffers (all: 25 mM Hepes-KOH pH 7.6 / 5% glycerol / 10 mM Mg(OAC)2) containing either 0.1 M K-glutamate, or 0.1 M K-acetate, or 0.1 M NaCl^?. The complexes were assembled on 1 kb linear DNA.
buffer:
1 wash+0.1 M K-glutamate
2 wash+0.1 M K-Ac
3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5

Changed:

<
<

buffer: 1 wash+0.1 M K-glutamate 2 wash+0.1 M KoAc^? 3 wash+0.1 M NaCl^?

>
>

Grids:
Nitrocellulose/carbon - 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain
1 - sample 1
2 - sample 2

Added:

>
>

3 - sample 3

Changed:

<
<

UrFormate^?: 0.75%, pH 4.5 Grids: 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain 1 sample 1

>
>

Nitrocellulose/carbon - 5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain
1B - sample 1
2B - sample 2
3B - sample 3

Deleted:

<
<

2 sample 2 3 sample 3

5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain 1B sample 1 2B sample 2 3B sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 615 Aug 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
- MSKCC
  - Dirk Remus
    - August 9, 2012
    - August 15, 2012

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

MSKCC

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Added:

>
>

Mcm2-7 - double hexamers around DNA >600kDa

buffer: 1 wash+0.1 M K-glutamate 2 wash+0.1 M KoAc^? 3 wash+0.1 M NaCl^?

UrFormate^?: 0.75%, pH 4.5 Grids: 3 uL sample, 15 sec glowdischarge w/Minghui program, 1min incubation, 30 sec stain 1 sample 1 2 sample 2 3 sample 3

5 uL sample, 20 sec glowdischarge w/Minghui program, 5min incubation, 30 sec stain 1B sample 1 2B sample 2 3B sample 3

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 515 Aug 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman
    - June 29, 2012
  - Steve Hubbard/Todd Miller
    - July 10, 2012
- MSKCC

Projects

NYU

Ned Seeman

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

July 10, 2012

Trip to Miller lab @ StonyBrook^?

MSKCC

Added:

>
>

Dirk Remus

August 9, 2012

Initial meeting

August 15, 2012

First 120kV session with JulienGros

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

Changed:

<
<

First 80kV session with John Maciejowski

>
>

First 80kV session with JohnMaciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 407 Aug 2012 - Main.EdEng

Contents

Projects
- NYU
  - Ned Seeman

Projects

NYU

Ned Seeman

Added:

>
>

June 29, 2012

Initial meeting

Steve Hubbard/Todd Miller

Added:

>
>

July 10, 2012

Trip to Miller lab @ StonyBrook^?

Deleted:

<
<

d19 3

MSKCC

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with John Maciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 307 Aug 2012 - Main.EdEng

Contents

Projects

Projects

Added:

>
>

NYU

Ned Seeman

Steve Hubbard/Todd Miller

d19 3

MSKCC

Prasad Jallepalli

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with John Maciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 207 Aug 2012 - Main.EdEng

Contents

Projects
- MSKCC

Projects

MSKCC

Changed:

<
<

July 23 2012

>
>

Prasad Jallepalli

Added:

>
>

July 10, 2012

Initial meeting

July 23 2012

First 80kV session with John Maciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Revision 123 Jul 2012 - Main.KenNguyen

Contents

Projects
- MSKCC
  - July 23 2012
    - July 23 2012

Projects

MSKCC

July 23 2012

Initial meeting

July 23 2012

First 80kV session with John Maciejowski

Ska proteins Ska1/2/3 wt - dimer - 180kDa Ska1/2 - monomer - <50kDa

buffer: Tris, pH 7.4, 150 mM NaCl^? , 1 mM EDTA, 1 mM DTT

First conditions:

Fresh Nitrocellulose/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
A1 - 2.5 ug/mL - Ska1/2/3 wt - Dark electron dense strands and poorly stained protein
A2 - 10 ug/mL - Ska1/2/3 wt
A3 - 25 ug/mL - Ska1/2/3 wt- Darker stained areas see oligomeric protein

Fresh Nitrocellulose/carbon - 25 sec glow discharge, 30 sec incubation, 30 sec stain
B1 - 2.5 ug/mL - Ska1/2/3 wt
B2 - 10 ug/mL - Ska1/2/3 wt
B3 - 25 ug/mL - Ska1/2/3 wt- Dark stain arease see oligomeric protein

Old formvar/carbon - 15 sec glow discharge, 30 sec incubation, 30 sec stain
C1 - 2.5 ug/mL - Ska1/2/3 wt
C2 - 10 ug/mL - Ska1/2/3 wt - globular looking protein
C3 - 25 ug/mL - Ska1/2/3 wt- GRID LOST

Old nitrocellulose/carbon - 20 sec glow discharge, 30 sec incubation, 30/45 sec stain
D1 - 2.5 ug/mL - Ska1/2/3 wt
D2 - 10 ug/mL - Ska1/2/3 wt
D3 - 25 ug/mL - Ska1/2/3 wt
D4 - 10 ug/mL - Ska1/2/3 wt

A5 - 10 ug/mL - Ska1/2/3 S34D - large oligomers
B5 - 10 ug/mL - Ska1/2/3 S34D
C5 - 10 ug/mL - Ska1/2
D5 - 10 ug/mL - Ska1/2 - monomers and oligomers

Old formvar/carbon - 20 sec glow discharge, 30 sec incubation, 60 sec stain
E1 - 10 ug/mL - Ska1/2/3 wt
E2 - 10 ug/mL - Ska1/2/3 S34D - poor staining, but area of oligomerization/aggregation?
E3 - 10 ug/mL - Ska1/2/3 S34D
E4 - 10 ug/mL - Ska1/2 - small globular monomers, but areas of oligomerization/aggregation?

Image stack

image #	Magnification	Grid	Construct
1-5	50kX	A1	Ska 1/2/3 wt
6-7	11.5kX	A1	Ska 1/2/3 wt
8-10	62kX	A1	Ska 1/2/3 wt
11-15	62kX	A3	Ska 1/2/3 wt
16-25	50kX	A3	Ska 1/2/3 wt
26-35	50kX	B3	Ska 1/2/3 wt
36-45	50kX	C3	Ska 1/2/3 wt
46-60	50kX	E4	Ska 1/2
61-70	50kX	E2	Ska 1/2/3 S34D
71-75	50kX	A5	Ska 1/2/3 S34D
76-80	50kX	D5	Ska 1/2
81-82	50kX	D2	Ska 1/2/3 wt

Set ALLOWTOPICVIEW =

Difference: CemEngProjects (1 vs. 24)

Revision 2410 Sep 2013 - Main.EdEng

Projects

NYU

Ned Seeman

June 29, 2012

Steve Hubbard/Todd Miller

February 23, 2012

March 9, 2012

April 6, 2012

May 4, 2012

June 12, 2012

June 20, 2012

July 10, 2012

August 21, 2012

September 26, 2012

November 07-08, 2012

June 2013

MSKCC

Dirk Remus

August 9, 2012

August 15, 2012

August 22, 2012

Prasad Jallepalli

July 10, 2012

July 23 2012

RCT

Revision 2324 Jun 2013 - Main.EdEng

Projects

NYU

Ned Seeman

June 29, 2012

Steve Hubbard/Todd Miller

February 23, 2012

March 9, 2012

April 6, 2012

May 4, 2012

June 12, 2012

June 20, 2012

July 10, 2012

August 21, 2012

September 26, 2012

November 07-08, 2012

June 2013

MSKCC

Dirk Remus

August 9, 2012

August 15, 2012

August 22, 2012

Prasad Jallepalli

July 10, 2012

July 23 2012

RCT

Revision 2218 Apr 2013 - Main.EdEng

Projects

NYU

Ned Seeman

June 29, 2012

Steve Hubbard/Todd Miller

February 23, 2012

March 9, 2012

April 6, 2012

May 4, 2012

June 12, 2012

June 20, 2012

July 10, 2012

August 21, 2012

September 26, 2012

November 07-08, 2012

MSKCC

Dirk Remus

August 9, 2012

August 15, 2012

August 22, 2012

Prasad Jallepalli

July 10, 2012

July 23 2012

RCT

Revision 2108 Nov 2012 - Main.EdEng

Projects