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=Polyacrylamide Gel on Brain Tissue Sections Test= ==Purpose== *Due to continued problem of tissue degradation in [[Matt:LabNotes/2016-1-20 | 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 *[[Matt:LabNotes/2016-1-20 | 2 sample dishes]] ==Experiment== *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 β100 uL dH20 #Prepare fresh 5% TEMED (in fume hood) ##2 uL TEMED β 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 #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== *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== *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
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