Berglund:Acrylamide

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