Berglund:Acrylamide

From OpenWetWare

Jump to: navigation, search

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
Personal tools