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

• • 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

August 15, 2012

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

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:

grids: nitrocellulose/carbon

Prasad Jallepalli

July 10, 2012

July 23 2012

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

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 =