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]
- Based on this general Matt:LabNotes/Polyacrylamide_Gel_Protocol
- Turn on UV lamp in AirClean hood for 15 minutes.
- Prepare fresh "AB mix" in a 0.5 ml pcr tube:
- 180 uL IEF 40% Acrylamide (made from powder)
- 20 uL Acrylamide / Bis (19:1; 38%:2%)
- 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.
- 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.
- 5 mg APS →100 uL dH20
- Prepare fresh 5% TEMED (in fume hood)
- 2 uL TEMED → 38 uL dH20.
- Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
- 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.
- 50 uL AB mix (FILTERED)
- 4 uL 10% BSA
- 138 uL dH20
- 4 uL 5% TEMED
- 4 uL 5% APS
- 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
- Sample A: Have a 22x22 coverslip over the 12x17 hole and add 100ul mix into corner of hole and then add 100ul mineral oil
- Leave in PCR hood for 30min (did not seal in bag)
- Aspirate mineral oil
- 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