Matt:LabNotes/2016-2-18: Difference between revisions

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>Mzcai
(Created page with "=Polyacrylamide Gel on Brain Tissue Sections with Argon Chamber Test= ==Purpose== *Use argon gas chamber (sealed plastic bag) to improve polymerization of polyacrylamide gel ...")
 
>Mzcai
Line 27: Line 27:
##4 uL                5% TEMED
##4 uL                5% TEMED
##4 uL                5% APS
##4 uL                5% APS
#Aspirate PBS from sample and then add 200ul gel-casting mix
#Aspirate PBS from sample and then add 150ul gel-casting mix
#Put in plastic bag and seal
#Put in plastic bag and seal
#Use vacuum to suck out air and then add argon gas
#Use vacuum to suck out air and then add argon gas
#Let sit in sealed bag for 30min
#Let sit in sealed bag for 30min
<!--
 
#Repeated everything for Sample B but let 138ul spread out completely over coverslip
 
==Results/Observations==
==Results/Observations==
*Sample A:
*Sample A:
**When picked up coverslip, some gel stuck to it
**It worked! >95% of gel mix polymerized
**After washing with TX-100 did not detect any gel on the glass
**I could aspirate the soluble mix that remained on top and the gel will get sucked on but won't detach from glass
**Did not seem to be able to wash away all the oil (formed droplets that couldn't be aspirated away with solution)
**Since I didn't believe it would work so well I did not spread it out evenly across glass
*Sample B:
*Sample B:
**After washing with TX-100 did not detect any gel on the glass
**It worked again!
**Same oil droplet problem as sample A
**There was actually a small droplet of polymerized gel from last time stuck to glass, picked it off
*PCR tubes of ~80ul leftover acrylamide mix after 30min:
**Some gel mix leaked under coverslip and polymerized, easily scraped away
**Tube with all mix sitting at the bottom was most polymerized but still not as stiff as jello, more goopy like melted tapioca
**Stuck nicely to glass when washing with 1X PBS
**Tube where mix was separated into droplets clinging to walls barely polymerized, the most polymerized was the largest portion stuck below the cap


==Conclusion==
==Conclusion==
*Mineral oil is too messy to wash away
*Using argon gas in plastic bag is needed for polymerization of thin gel on coverslip
*Polymerized gel is much less solid than I expected
*Gel sticks to glass well
**Did we make the right A/B mix? Enough Bis-acrylamide?
*Still need to test if dye-probes can diffuse to tissue and be imaged clearly
**Maybe it was exposed to too much air and couldn't polymerize completely
*Still need to test making thinner gel
***The polymerization in tube would suggest that
*Then need to test whether can do DARTFISH with gel right after or during fixation
**Maybe the gel in Sample B was so liquid-like it got aspirated along with oil-->

Revision as of 02:48, 19 February 2016

Polyacrylamide Gel on Brain Tissue Sections with Argon Chamber Test

Purpose

  • Use argon gas chamber (sealed plastic bag) to improve polymerization of polyacrylamide gel
  • Creating a low density polyacrylamide gel surrounding the tissue can keep tissue together while allowing diffusion of DNA oligos and enzymes
  • Previously tried minimizing oxygen exposure (which inhibits polymerization) by using vacuum chamber
  • Sample dish A

Experiment

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Use "AB mix" from last week:
    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 AB mix into a 1.5 mL microcentrifuge tube.
  4. Degas the AB mix by using vacuum and sonicator
  5. Prepare fresh 5% APS (ammonium persulfate)
    1. 5 mg APS →100 uL dH20
  6. Prepare fresh 5% TEMED (in fume hood)
    1. 2 uL TEMED → 38 uL dH20.
  7. Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
  8. Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels
    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
  9. Aspirate PBS from sample and then add 150ul gel-casting mix
  10. Put in plastic bag and seal
  11. Use vacuum to suck out air and then add argon gas
  12. Let sit in sealed bag for 30min
  1. Repeated everything for Sample B but let 138ul spread out completely over coverslip

Results/Observations

  • Sample A:
    • It worked! >95% of gel mix polymerized
    • I could aspirate the soluble mix that remained on top and the gel will get sucked on but won't detach from glass
    • Since I didn't believe it would work so well I did not spread it out evenly across glass
  • Sample B:
    • It worked again!
    • There was actually a small droplet of polymerized gel from last time stuck to glass, picked it off
    • Some gel mix leaked under coverslip and polymerized, easily scraped away
    • Stuck nicely to glass when washing with 1X PBS

Conclusion

  • Using argon gas in plastic bag is needed for polymerization of thin gel on coverslip
  • Gel sticks to glass well
  • Still need to test if dye-probes can diffuse to tissue and be imaged clearly
  • Still need to test making thinner gel
  • Then need to test whether can do DARTFISH with gel right after or during fixation