single strand DNA production

Ashok Aiyar axa12 at po.CWRU.Edu
Tue Mar 19 15:34:09 EST 1991


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 at cwbio.bio.cwru.edu
aiyar at cwru.cwru.edu
axa12 at po.cwru.edu

Thank you,

Ashok Aiyar
-- 
Ashok Aiyar
axa12 at po.cwru.edu
aiyar at cwbio.bioc.cwru.edu



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