r.james at uea.ac.uk
Wed Nov 9 03:38:59 EST 1994
> bawagan at umdnj.edu (Hinayana Bawagan) writes:
> > One of two methods I will use for mutagenizing my plasmid DNA is by
> >hydroxylamine. I have no experience with this method but it has been
> >successful for isolating temperature sensitive mutants of yeast.
> > The protocol that I have states that the mutagenizing solution should be
> >prepared FRESH. I would like to ask if this is a strict caveat and what is
> >the reason for it. The protocol that I have appears straightforward and easy.
> >To those who have experience with the method, did you have problems, tricks,
> >precautions that you would like to share with me?
We incubate the hydroxylamine with dsDNA at 70C to promote localised
opening of the helix. After incubation for 0,60,120,180,240 and 300
mins we then precipitate the DNA, resuspend in TE buffer and transform E.coli. If you count the number of transformant colonies
for each time point and then plot the number as a % of the number of
transformants at 0 time point, you can see which time point gives
about 1%. This appears to be the optimum for mutagenesis.
Hydroxylamine mutagenesis will only give G:C to A:T transitions, so
you will not get all possible mutations. To get around this we use a PCR mutagenesis protocol which incorporates dITP in the PCR reaction.
The reference is Spee, de Vos and Kuipers Nucl. Acids Res. 21,
Good luck with the mutagenesis.
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