The fractionation of ribonucleic acid on a preparative scale by polyacrylamide gel electrophoresis

Volume 1, number 5 rent dropped sharply during a run_ This was ffund to be due to tie relatively high electrical resistance across tie @ass rixmbrane in tie floor of the eiution chamber; t&s caused local heating resuXng 31 bubble fa3mation in tile eiution chamber ar.d pariicular~y in the membrane itself, which in turn increased the resistance still further.This dSEcul?y was overcome by nisirig &he buffer concentratior~ in the 53wer reservoir and membrane holder to ?en times the standard concentration.The gel was made up in the standard buf &I; this was also used for the top reservoir and for du?ion.Gels were normally polymerized in the apparatus at room temperature (2lT.).After polymerization, cooling water was pumped though the jack& until the entire experimerrt had been completed.The RNA sample was dissolved in standard electrophoresis bufier containing 5% sucrose (w/v) usually in a vohnrne of approximately 5 ml and was careftiy layered on top of the gel after the apparatus had been set up and the buffer compartments filled.With the Buckler power pack set for constant current, a cutient of 200 ma was used, giving an applied voltage of 3 30 V between the upper, cat+&, compzrtment and tie lower.anode, compartment.il.13 dution bufffx flow rate of appr0xG mate& 3 mllm was fund to be satisfactcry.Water a: 3°C was pumped througl~ the water-jacket of the apgamtus from a wa?er bath ke$ cool by a Grant CC 35 -I Coder Unit.The~eluate w& monitored w@ a& l&3 Gticord 33 tid was reqrded on a' yokogawa c&art re-_ .-,FQ_ 2a.Joyce Loebl Ckromoscan record of analytkal PO&auylarnide ge3 electrophoretogmm oi moud retidocyte RNA.50 ~g RNA on a 2.4% gel, e?ectrophoresis ior 145 hr at 4T, potential dXference IQ Vfcm, 4Fluient 3.5 ma.Gel scanned at 265 mu.2b.Sucrose detity gradient of mou!x zeticrjc~fie RNA_ 175 pg RNA 3ayerrd on S-20% SUMSC gradient, centrZuged 2 hr, 30 min in 3 X 5 d swing out rotor, MSE superspced 50 ultracentxifbge at 39 ODD rpm.Gm&ert analysed using a flow cell with a U&an3 S? 800 spectrophotometer.