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Denaturing Urea PAGE -Small Gel 1. Prepare denaturing polyacrylamide gel solution. Use Gibco/BRL apparatus.
 

Summary: 31
Denaturing Urea PAGE - Small Gel
1. Prepare denaturing polyacrylamide gel solution. Use Gibco/BRL apparatus.
7.2 % 9.6 % 12 %
10X TBE 2.5 mls 2.5 mls 2.5 mls
Urea (ultrapure) 10.5 g 10.5 g 10.5 g
40% Acrylamide 4.5 mls 6 mls 7.5 mls
ddH2O 10.5 mls 9 mls 7.5 mls
Total Volume 25 mls 25 mls 25 mls
Use 40% acrylamide stock for DNA/RNA gels. Do not use 30% stock for SDS-PAGE gels. Mix in 50
ml beaker. Heat gently in microwave (~ 15 seconds) to dissolve urea. Syringe filter (0.45 m)
gel mix into sidearm flask and degas for 5-10 minutes.
If urea is not ultrapure grade, deionize as follows: Add 0.05 g AG501-X8(D) resin. Mix at room
temperature for 5 minutes. Spin in IEC for 5 minutes at 2000 rpm.
2. Pour gel. Add 25 l TEMED and 50 l 25% APS. Pour gel to ~ 0.5 cm from top. Insert clean, dry
comb at an angle to prevent trapping of bubbles. Push all the way down, but don't trap any bubbles.
3. Pre-electrophorese gel. Use 1 X TBE in upper and lower reservoirs. Remove comb. Run at 20
watts for 15 minutes. Warmth plus urea denatures nucleic acids.
4. Denature samples. To RNA, add an equal volume of sample buffer (100 l formamide + 1 l 0.5 M
EDTA, pH 8 + 1 l 100X BPB). Heat for 1 minute at ~95C (tubes in steam over boiling water in

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine