Berglund:Acrylamide
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For an excellent review on acrylamide and problems associated with acrylamide gels (native and denaturing) please refer to:
www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf
10% RNA Denaturing Polyacrylamide Gel
Materials
Total volume: 75ml
- 18.75ml of 40% 19:1 acrylamide:bis acrylamide
- 7.5ml 10xTBE buffer
- 31.5g of Urea (fc=7M: 60MW* 0.075L*7M)
- add water to 75ml
2x denaturing dye:
- 200µl 1% Bromophenol blue
- 200µl 1% xylene cyanol
- 500µl formamide
- 100µl 10x TBE
Glass plates: Need (1) 8 inch x 6 1/2 inch glass plate plate and (1) 7 inch x 6 1/2 inch glass plate (each 1/8 inch thick)
teflon spacers and combs that are .75mm thick
Methods
This is for a small gel:
- Place spacers between plates, clamp with clamps making sure spacers touch each other
- Mix 150µl AP and 25µl of TEMED into the premade gel base
- Pour
- Place comb into plate. Wait for gel to polymerize (30 min)
- Remove bottom spacer from plates
- Place into gel apparatus and tighten down
- Fill reservoirs with 1XTBE buffer
- Blow out bottom of gel where spacer was with bent needle to remove bubbles, blow out wells carefully
- Run at 400V-450V for 30 min or until gel warms up
- While gel is running prepare the samples
- Add 5µl of dye to 5µl of sample
- Add 5µl of dye to 5µl RNA standards of your choice
- Denature sample and standards for 5 min at 95° C
- Stop running gel (no voltage should go through the gel while you are loading)
- Blow out wells with straight needle to remove urea
- Load samples
- Run at 200V-450V until you are satisfied (sometimes longer sometimes shorter)
- Remove gel from apparatus