Michael S. Parmacek
1) 40% Bis-acrylamide (20 g acrylamide and 1 g bis-acrylamide in 50 ml ddH20)
2) 10X oligo TBE, pH 8.8- 1M Tris (MW 121.1)
51.3 g Boric acid
3.72 g EDTA (tetrasodium salt)
H20 to 1L, adjust pH to 8.8 if necessary
3) Running Buffer- 80% formamide, 10 mM NaOH, 0.1% Bromphenol blue
1) Prepare 20% Bis-acrylamide-7M urea gel:
50 ml 40 % Bis-acrylamide solution
42 g urea
10 ml 10X oligo TBE, pH 8.8
1 ml 10% APS
25 ul temed
adjust volume to 100 ml with ddH20
use 1.5 mm spacers and 6-8 well comb, use 1X oligo TBE as Buffer
let gel set at least 30' to polymerize. Pre-run the gel for 15 minutes prior to loading.
2) Resuspend deprotected synthetic oligonucleotide in 30 ul of ddH20 and quantitate the concentration by measuring E260/280 (for oligos 1 OD = 20 ug/ml)
3) Add the oligo to 60 ul of Running Buffer (total volume 90 ul). Boil each sample 3 minutes and place on ice prior to loading on gel.
4) Load samples on the 20% Bis-acrylamide-7M urea gel and run at 300 Vuntil the dye front is 1/2 to 3/4 of the way down the gel.
5) Visualize the band with Kodak-TLC sheet under short-wave UV light.
6) Cut the band out of the gel with a razor blade. Mince the gel slice and place in a microfuge tube. Add 500 ul of 0.3 M Na Acetate, pH 5.2/1 mM EDTA solution to the tube and elute oligonucleotide from the gel at 37°C with gentle shaking overnight.
7) In the AM, add two volumes of EtOH to the eluant and precipitate on dry ice for 1-2 hour. Spin in microfuge 15', 4°C to collect the oligonucleotide.
8) Wash once with 1 ml of 70% EtOH. Dry down oligo in the Speed-vac.
9) Resuspend the oligo in the desired volume (usually 30 ul initially) and solution (usually H20) and re-quantitate its concentration.