Northern blotting of RNA denatured in glyoxal without buffer recirculation.
A rapid and easy procedure for preparing Northern blots is described. RNA is denatured with glyoxal in the presence of ethidium bromide and glycerol, then electrophoresed through agarose in a buffer that does not require recirculation. Without any additional washes, the RNA is vacuum-blotted to a nylon membrane in NaOH, which simultaneously removes the glyoxal adducts. All of these steps plus prehybridization of the filter and addition of a digoxygenin-labeled probe can be completed in one day. Using standard procedures to wash the filters and detect the probe, the entire procedure can be completed within two days.
Burnett
W V
WV
Bristol-Myers Squibb, East Syracuse, NY 13057-5050, USA. wburnett@usccmail.uscc.bms.com
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D002478:Cells, Cultured; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D006037:Glyoxal; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D012984:Software
D015342:DNA Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006037:Glyoxal; D012313:RNA; D004996:Ethidium