Polyacrylamide Gel Electrophoresis of Oligonucleotides

1.Pourandpolymerizea20%polyacrylamidegel,noUrea.2.Removeclamps.Rinsewithwater.Removecomb.Rinsetopofgelwell.3.Insertcombteethdownuntilteethjusttouchthegel.Mounttheplatesinapparatus.Fillchamberswith0.5XTBEandprerunatlowwattagefor10min.4.Loadoligonucleotidestogel.5.Runnarrowgelsat35W.Runwidegelsat80W.RununtilBromophenolBlueis3/4waydown.6.Decantbufferfromapparatus.Removegelandlayflat.7.Removecombandsidespacers.Pryapartplateswithaspatula.Ifthegelisstucktothetopplate,resettleplatesandinvert.8.Lifttheupperplateoff.Ifbubblesarose,wetgelwith10%AceticAcidandworkthemout.PatdrygentlywithaKim-Wipe.9.If35Sor33Pnuclidesareused:A.Soakgel15minin10%Aceticacid.Patdry.B.CovergelwithWhatman3MMpaper.Liftoffgelstuckonpaper.Coverwithplasticwrap.C.Dryinvacuumgeldrierfor20minat80C.Ifsoaked,dry60min.D.Ifsoaked,removedplasticwrap.E.ExposetoX-rayfilm.10.Ifnon-rADIoactive,stainwithEthidiumbromidefor5min.Illuminatewithultraviolet