Filament Set-up Procedure:

• 1. Cut a piece of Tungsten wire 4-5 cm (0.15 mm diameter, I need double check it).
• 2. Wearing gloves, straighten the wire, swipe it with emery cloth and clean it with an ethanol soaked kimwipe.
• 3. load the filament into the metal holder.

Rotary Stage Set-up Procedure:

• 1. Clean stage and motor plate with scrub pad and ethanol with an ethanol soaked kimwipe.
• 2. Place small strips of double stick tape on the stage to hold the mica.
• 3. Label the stage to indicated sample position.
• 4. Place filter paper on motor plate with bent corner facing the filament (this is your indicator).

Placing the Rotary Stage, Filament and Carbon Gun in the Vacuum Chamber of the Edwards:

• 1. Place the rotary stage in the center of bell jar.
• 2. Attach the electric cable to the electric feed trough.
• 3. Turn on the power and check to make sure the table will rotate.
• 4. Wearing gloves, place the carbon/metal gun in position over the rotary table.
• 5. Wearing gloves, place the filament between the two filament posts. Adjust the height of the filament to give a predetermined shadow angle (we set up filament 8 cm away from rotary table center and 1 cm higher than rotary table).

Prepare Samples for Shadow:

• 1. Chromatin samples was fixed by 0.6% glutaraldehyde and passed through Gel filtration column (A-5 or Sepharose Cl-4B)
• 2. The purified chromatin sample (10 l) was mixed with 20X spermidine buffer (0.5 l) and mounted to glow-charged carbon-coated grid at RT for 1-2 min.
• 3. The sample was dehydrated by ddH2O/ethanol series ( ddH2O for 30 sec; ddH2O for 4 min; 25% ethanol for 4 min; 50% for 4 min; 75% for 4 min and 100% ethanol for 4 min)
• 4. Put the grids on the rotary table and mark the order of samples before start shadowing.

Tungsten Shadow:

• 1. Wait to achieve vacuum of ≤2 x 10^-6
• 2. Put the low tension selector (LT ) to filament position, and put the voltage knob to B and D.
• 3. Have protective eye goggles ready. Never look directly at the source without eye protection.
• 4. Turn on the rotary stage.
• 5. Turn on the power to low tension supply.
• 6. Start stopwatch and dial up current to 18%. Wait approximately 30 seconds (the filament should glow red and there will be a slight loss of vacuum due to outgassing).
• 7. Slowly increase current until you reach 24% and stay at 24% for 2 min and 10 seconds.
• 8. Then rapidly turn current down and shut low tension selector off.
• 9. Seal and Vent chamber and remove samples.
• 10. Remove the filament and rotary stage from the bell jar.
• 11. Follow shut down procedures for the Edwards Vacuum Evaporator.
• 12. Clean the equipment and return everything to its proper location.

Sublevel topic

subsub level topic

• Pick appropiate metal diameter

• Pick LT or HT on Selector
  • LT 10V 90 Amp
  • HT 30V 30 Amp
  • Amp meter reads percentages rather than straight Amps