Plasmid sequencing

Sasha Kraev bckraev at aeolus.ethz.ch
Wed Nov 9 15:20:27 EST 1994


I use the procedure of H.Voss et al (Meth.Molec.Cell.Biology, 3:153-155(1992))
which is similar to their earlier paper (Nucleic Acid Res.,18:1067 (1990)).
However, I use 33P dATP as label and control denaturation/neutralization by
adding phenol red dye. Can post details, if required. USUALLY I have (great)
success, but there are some clones that just dont sequence with this protocol
(and I guess with any other using T7 polymerase), because of their unusually
low copy number. Could it be that you have got one of those? I tried some time
ago to raise a discussion on the matter of strain/clone effect on template 
quality, but it seemed that nobody was interested. Hope this helps, Sasha



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