Matt:LabNotes/2016-2-9

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Polyacrylamide Gel on Brain Tissue Sections Test[edit]

Purpose[edit]

  • Due to continued problem of tissue degradation in grey matter of brain tissue need to try methods to keep tissue whole during DARTFISH process
  • Creating a low density polyacrylamide gel surrounding the tissue can keep tissue together while allowing diffusion of DNA oligos and enzymes
  • 2 sample dishes

Experiment[edit]

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Prepare fresh "AB mix" in a 0.5 ml pcr tube:
    1. 180 uL IEF 40% Acrylamide (made from powder)
    2. 20 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 AB mix (FILTERED)
    2. 4 uL 10% BSA
    3. 138 uL dH20
    4. 4 uL 5% TEMED
    5. 4 uL 5% APS
  8. Aspirate PBS from both samples and then add 100ul to each of the two samples
    • Sample A: Have a 22x22 coverslip over the 12x17 hole and add 100ul mix into corner of hole and then add 100ul mineral oil
      • Hard to tell if the acrylamide covered the whole surface before adding mineral oil
    • Sample B: No coverslip, just add 100ul mix on top to cover whole surface then added 400ul mineral oil to cover the whole surface
      • No clear separation between mix and oil but likely due to how thin the layer
  9. Leave in PCR hood for 30min (did not seal in bag)
  10. Aspirate mineral oil
  11. Wash with 0.25% TX-100 in H2O twice to remove majority of oil

Results/Observations[edit]

  • Sample A:
    • When picked up coverslip, some gel stuck to it
    • After washing with TX-100 did not detect any gel on the glass
    • Did not seem to be able to wash away all the oil (formed droplets that couldn't be aspirated away with solution)
  • Sample B:
    • After washing with TX-100 did not detect any gel on the glass
    • Same oil droplet problem as sample A
  • PCR tubes of ~80ul leftover acrylamide mix after 30min:
    • Tube with all mix sitting at the bottom was most polymerized but still not as stiff as jello, more goopy like melted tapioca
    • Tube where mix was separated into droplets clinging to walls barely polymerized, the most polymerized was the largest portion stuck below the cap

Conclusion[edit]

  • Mineral oil is too messy to wash away
  • Polymerized gel is much less solid than I expected
    • Did we make the right A/B mix? Enough Bis-acrylamide?
    • Maybe it was exposed to too much air and couldn't polymerize completely
      • The polymerization in tube would suggest that
    • Maybe the gel in Sample B was so liquid-like it got aspirated along with oil