Sample prep

Goal

Prepare negative stain grids for TEM to assess particle quality, general topology or obtain a low-resolution dataset.

Background

A complex of biological molecules (generally >50kDa) is deposited onto plasma-discharged carbon film grids. Plasma-cleaning is a means of ensuring that the surface of the carbon is uniformly hydrophilic and will allow your sample to stick to the surface of the carbon. After deposition or your biological sample onto the carbon film, uranyl acetate or formate is used to coat the rest of the film with uranium atoms. These extremely dense atoms do not allow transmission of electrons as well as your biological sample. This creates a negative staining of your sample, with great contrast between it and the uranium atoms.

Making 2.5% uranyl formate solution (enough for 4 grids)

  1. Mass out 25mg of UF in an eppi-tube

  2. Wrap in tinfoil

  3. Add 1mL of boiled and cooled (to remove carboxylic acid) dH20

  4. Invert to dissolve most of the UF

  5. Add 2uL of KOH to the lid of the eppi-tube (adding it directly will cause precipitation of UF)

  6. Invert again until the rest is dissolved

  7. Filter into a new eppi-tube wrapped in tinfoil

Discharging grid

Use CF400-CU UL (or similar) grids, do not use formvar grids.

  1. Load the 20nm carbon recipe on the plasma cleaner

  2. Vent the chamber to open

  3. Place grids in grid holder

  4. Place grid holder in middle (third hole) of stage

  5. Replace door and Run Recipe

  6. Watch for plasma discharge

  7. Vent chamber to open

  8. Take out grids, replace door

  9. Pump Down chamber to ~30mTor

  10. Finish Up to place in standby

Staining

If sample has >10% glycerol, wash sample in non-glycerol buffer after sample application (and incubation time), but before first UF staining.

  1. Pick up charged grid (dull side up) with clamping tweezers

  2. Apply 4uL of sample onto surface of carbon, without touching pipette to grid

  3. Incubate sample for ~30s

  4. Line up 5x35uL drops of UF on parafilm

  5. After incubation, apply carbon side of grid to first UF drop, using surface tension to soak grid from only carbon side

  6. Blot immediately to remove UF crystals

  7. Use same surface tension to carbon approach to move through each of the next 4 drops of UF

    • Swirl and spend 10s in each drop

    • Do not blot between drops

  8. Blot after applying sample to the last drop

    • Be thorough with blot, but leave thin layer

    • Also blot between tweezer tips

  9. Allow sample to dry ~30s before depositing in box

  10. Dispose of filter paper and tips in radioactive waste, if contaminated with UF

  11. Repeat for susequent grids

Clean up

  1. Pipette unused UF onto parafilm

  2. Soak up UF with filter paper, dispose in radioactive waste

  3. Ball up parafilm, dispose in radioactive waste

  4. Change absorbant pad, if contaminated

  5. Lock up radioactive waste

  6. Return all tools to storage places

  7. Fill out radiation log book (located on top of plasma cleaner)

  8. Fill out radiation waste log (located on top of radioactive waste bucket)