Berglund:Acrylamide

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

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