Matt:LabNotes/Polyacrylamide Gel Protocol

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Polyacrylamide Gel Protocol

Purpose

  • Make gel to cover brain tissue section for DARTFISH to prevent tissue degradation


Materials

Protocol

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Prepare fresh "ABD mix" in a 0.5 ml pcr tube:
    1. 90 uL IEF 40% Acrylamide (made from powder)
    2. 10 uL Acrylamide / Bis (19:1; 38%:2%)
  3. Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the ABD mix into a 1.5 mL microcentrifuge tube.
  4. Prepare fresh 5% APS (ammonium persulfate). 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. 5 mg APS →100 uL dH20
  5. Prepare fresh 5% TEMED (in fume hood)
    1. 2 uL TEMED → 38 uL dH20.
  6. Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
  7. 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. 138 uL dH20
    4. 4 uL 5% TEMED
    5. 4 uL 5% APS