diaz@aecom.YU.EDU (Daniel Diaz) (11/05/87)
In an attempt to avoid the complications of probe renaturation during S1 nuclease mapping of transcription start sites, I wish to easily isolate single-stranded DNA from labeled duplex. The new Guide to Molecular Cloning Techniques put out by our friends at Methods in Enzymology has not been helpful in this regard. Although helpful, I've found the book in need of excision repair. Electrophoresis in polyacrylamide, 7M urea has been used by some, but I don't know how well it works. Recloning into m13 would be easiest, but time-consuming. Please send any suggestions via email. -- dn/dx Dept Molecular Biology diaz@aecom.yu.edu Dan Diaz AECOM "I've met Craig Werner"
Mitchell_K_Hobish@cup.portal.com (11/10/87)
Have you considered cesium chloride (or the equivalent) ultracentrifu- gation? It used to work well for me when isolating polyoma or SV-40 DNA in supercoiled, open circle, nicked, etc. forms... Mitchell_K_Hobish@cup.portal.com CIS: 70320,257 MCI Mail: MHOBISH GEnie: MitchH DELPHI: MHOBISH
diaz@aecom.YU.EDU (Dizzy Dan Diaz) (11/13/87)
In article <1370@cup.portal.com>, Mitchell_K_Hobish@cup.portal.com writes: > Have you considered cesium chloride (or the equivalent) ultracentrifu- > gation? It used to work well for me when isolating polyoma or SV-40 > DNA in supercoiled, open circle, nicked, etc. forms... CsCl does not quite suit the purpose I had in mind. I was interested in separating strands of 5' labelled DNA frgaments < 1kb long. CsCl by itself would be unsuitable for large-scale preparations anyway since CsCl it nondenaturing. Alkaline gradients would be best for that purpose. Thanks for the suggestion anyway. -- dn/dx Dept Molecular Biology diaz@aecom.yu.edu Dan Diaz Albert Finkelstein College of Quackery