Daniel:Notebook/RNAFACS/2015-11-24

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Revision as of 19:26, 24 November 2015 by >Djacobse (→‎Gel Casting)
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PAGE Gels on Slides-Trial

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Gel Casting

  1. Remove 8 Bind-Silane treated slides from storage box in dessicator and place face up in AirClean hood.
  2. Turn on UV lamp in AirClean hood for 15 minutes.
  3. Label slides with numbers and date using a SHARPIE pen (other inks will wash off in hexane)
  4. Almost completely cover slides with coverslips (Fisherbrand, 18mm x 30mm, #1, untreated). Keep a small area of oval exposed for subsequent gel loading.
  5. Prepare fresh "ABD mix" in a 15 ml polystyrene conical:
    1. 9 mL IEF 40% Acrylamide
    2. 1 mL Acrylamide / Bis (19:1; 38%:2%)
  6. Using a 3-cc syringe and a 0.22 micron filter, filter ~1 mL of the ABD mix into a 1.5 mL microcentrifuge tube.
  7. Prepare fresh 5% APS. Notably, the APS bottle should be stored in some sort of room-temperature dessicator. We just store it inside a large plastic screw-top container.
    1. 0.5 g APS →10 mL dH20
  8. Prepare fresh 5% TEMED
    1. 2 uL TEMED → 38 uL dH20.
  9. Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides.
    1. 50 uL A-B-D mix (FILTERED)
    2. 4 uL 10% BSA
    3. 135 uL dH20
  10. Split reaction mix into 2 tubes (95 uL per rxn) and add to each:
    1. 2 uL 5% TEMED
    2. 2 uL 5% APS
    3. X uL Template (see reaction table)
  11. Suck up 18 microliters of the mix and pipet it into the small exposed area of the oval, such that surface tension pulls the liquid into the space between the coverslip and glass to cover the full surface area of the oval. The liquid should distribute under the coverslip such that only a small amount (1 to 2 uL) cannot fit. Slide the coversip over such that the oval is completely covered.
  12. Place the slides on a flat tray and load the tray into the argon chamber (and fill with argon). Allow gels to polymerize for ~30 minutes.
  13. Remove slides from argon chamber. Use a razor-blade cleanly remove cover-slips from polymerized gels (just stick the edge of the blade under the coverslip and gently "pop" it up)
  14. To wash off the excess acrylamide monomer, place slides in a dH20 filled plastic Coplin jar and incubate for 30 minutes at RT with slow shaking.
  15. Remove slides from Coplin jar and place face up in PCR hood.

Reaction Table

  Reaction 1 Reaction 2
Base Nuclei (total) 5.25E+05 5.25E+05
Base Nuclei ( per uL) 1.75E+04 1.75E+04
Dilution 1:20 1:10
Template Nuclei (per uL) 875 1750
Reaction Volume 2.5 5
Reaction Concentration (N/uL) 21.9 87.5
Total Nuclei in Gel 394 1575

RNA Scope