axa12@po.CWRU.Edu (Ashok Aiyar) (03/20/91)
Hello: I have my insert cloned into pBluescriptKS+. I am currently trying to make ssDNA for mutagenesis. The procedure that I use seems to me to extremely in efficient. It involves superinfecting with M13KO7. Thi is followed by PEG/NaCl precipitating the phage from the bacterial supernatent, followed by loading it on a CsCl gradient. After pulling the bands and precipitating them (after dialysis), I find that I have recovered mayber 5 micrograms ssDNA (from a one liter culture), while the bulk of the band was helper phage. This is turning out to be extremely anoying. I have also tried Qiagen columns. My recoveries as visualized by gel electrophoresis appear to be much better, but the DNA is really dirty )(when I try to sequence with it). The specific areas the that I would like some clarification on are: a) the optimal titer of infecting helper phage. b) the optimal density of the culture that is infected. c) any hints on phage recovery before and after the gradient. d) any protocols for ssDNA production by some other method such as Qiagen, that has worked well. Suggestions and replies can either be posted on this bulletin board, or mailed to me at: aiyar@cwbio.bio.cwru.edu aiyar@cwru.cwru.edu axa12@po.cwru.edu Thank you, Ashok Aiyar -- Ashok Aiyar axa12@po.cwru.edu aiyar@cwbio.bioc.cwru.edu