See general guidelines for metal shadowing here.

Rotary shadowing using the Edwards Auto 306 Evaporator

General setup

• Make sure gloves are worn at all times and that surfaces are cleaned with EtOH^? and a Kimwipe to reduce oils/residues during procedure.
• Turn on Edwards Evaporator and start Turbo.
• Vent the chamber, make sure the system is tripped and that there is no current running through the system.
• Attach rotary stage attachment and connect it to the evaporator. Turn on to make sure it can rotate.
  • To increase the platform, double-sided carbon tape and the top of a small petri dish may be used.
• Attach filament holder attachment to leads B & D.
• Position the attachments such that the rotary stage platform is 8cm away and 1cm down from the filament.

Set-up for rotary shadowing

Protein rotary shadowing

Pt rotary shadowing of TMV on formvar/carbon coated copper grids

Filament set-up

• Cut a piece of Tungsten wire (3-5cm long and 0.15 mm diameter) to fit into the filament holder.
• Cut a 2.5 cm pice of platinum (Pt) or Pt/Pd wire.
• Clean both wires with EtOH^?/Kimwipe
• Wrap the Pt wire around the middle of the tungsten wire.

Protein spreading

• Glow discharge in air or amylamine a formvar/carbon coated grid as per standard procedures.
• Add protein sample to grid similarly as one were to negatively-stain the sample.
• If the buffer contains high salt or other components that may interfere with shadowing, blot away protein sample and wash in buffer or H2O. Additionally wash grid in EtOH^? in several steps increasing EtOH^? to 100% (e.g. 25%, 50%, 75%, 100% EtOH^?).
• Blot dry with filter paper.

Pt shadowing

• Place grids on rotary stage and start cycle.
• Wait for vacuum to be below 2x10-6 mb. With LN2 and overnight pumping can reach ~2x10-7 mb.
• Wear protective eye wear and never look directly at the filament.
• Reset circuit and turn selector to B&D with LT.
• Start rotation of stage. Make sure grids can comfortable stay on platform.
• Slowing increase current to outgass metal making sure vacuum remains <2x10-6.
• When stabilized slowly increase current to ~50% and the Pt wire should melt and form a ball in the center.
• Let shadow for 1-5min to evaporate the Pt. At this point the vacuum will decay and recover. Slowly increase current <75% allowing vacuum to recover and all Pt to evaporate.
  • If you ramp the current too fast the tungsten wire can melt and break.
• Turn off power to filament, trip circuit and vent chamber.

DNA rotary shadowing

W rotary shadowing of pUC19 DNA

Filament set-up

• Cut a piece of Tungsten wire (3-5cm long and 0.15 mm diameter) to fit into the filament holder.
• Clean wires with EtOH^?/Kimwipe

DNA spreading

• Glow discharge in air or amylamine a formvar/carbon coated grid as per standard procedures.
• Add DNA sample to grid around 5ug/mL-25ug/mL. Dilute in PBS with 20 mM MgCl2.
• If the buffer contains high salt or other components that may interfere with shadowing, blot away sample and wash in H2O. Additionally wash grid in EtOH^? in several steps increasing EtOH^? to 100% (e.g. 25%, 50%, 75%, 100% EtOH^?).
• Blot dry with filter paper.

W shadowing

• Place grids on rotary stage and start cycle.
• Wait for vacuum to be below 7x10-7 mb. With LN2 and overnight pumping can reach ~2x10-7 mb.
• Wear protective eue wear and never look directly at the filament.
• Reset circuit and turn selector to B&D with LT.
• Start stage rotation.
• Slowing increase current to outgass metal making sure vacuum remains <1x10-6.
• When stabilized slowly increase current to 30%.
• Let shadow for 1-5min to evaporate the W. At this point the vacuum will decay and recover. Slowly increase current <75% allowing vacuum to recover and and maintaining vacuum <2x10-6mb.
• Turn off power to filament, trip circuit and vent chamber.

• Set ALLOWTOPICVIEW =