ssDNA prep for mutagenesis only getting helper phage back
forrest at biosci.uq.oz.au
Wed Nov 16 23:12:39 EST 1994
Another prob to throw at you. I've been using the Biorad, Muta-gene
phagemid kit to try to do some mutagenesis, it uses the f1 origin of
replication on the phagemid to be mutagenised and a helper phage with
kanamycin resistance. The problem is that all I seem to be getting back is the
helper DNA rather than the ss phagemid. We thought that it might be the
kanamycin was off but as the kan resistance is on the helper this seems unlikely
with the large yield of helper dna.
The kit suggests it could be something to do with the insert sequence
and the phagemid used, in this case I'm using pGEM-T (pGEM5), with a 1.5kb
If anyone has had similar problems and found a way of getting over it,
please feel free to give me an email :).
Another thing was the cultures prior to infection with the helper took
ages to get up to an OD600 of 0.3. We attributed this to thef act that there
was both chloramphenicol and ampicillin in the media.
Thanks for any suggestions,
Alistair Forrest (Struggling PhD student)
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