Filter transfer of genomic libraries in a state accessible to DNA-binding proteins.
I have developed a method for transferring plaque DNA of lambda genomic libraries onto 3MM filters in a state accessible to DNA-binding proteins. DNA bound to 3MM is available to proteins as large as Escherichia coli RNA polymerase and maintains template activity similar to that in free solution. Lambda Plaques can be lifted onto 3MM filter disks, deproteinized, and used for transcription assays in vitro. The RNA synthesized is complementary to phage rather than to E. coli DNA and plaques can be identified by autoradiography. Furthermore, the filters can subsequently be probed with radioactive nucleic acids under standard hybridization conditions. Finally, colorimetric assays can be employed with lactate dehydrogenase (LDH) A in which plaques are identified by the localized reduction of nitroblue tetrazolium.
D000818:Animals; D001345:Autoradiography; D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000201:enzymology; D007770:L-Lactate Dehydrogenase; D009693:Nucleic Acid Hybridization; D051381:Rats; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D004247:DNA; D007770:L-Lactate Dehydrogenase; D012321:DNA-Directed RNA Polymerases