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(NON) muatgenesis

Mon Jul 25 14:23:00 EST 1994

Hello Methods/reagents people-

I am seeking the voices of experience (no TGIF here)!!!
I'm trying to perform site-directed mutagenesis by Kunkel's
method.  I am afraid of errors with PCR based methods, and having
to sequence kB's of DNA.  There arwe not many unique sites that i
can use to target a small DNA fragment.  The mutants are single or
double amino acid substitutions OR deleting 100 amino acids (300
nuc.).  I assume the DNA contains uracil since the transformation
efficiency in CJ236 os 10^5 better than on MV1190. The single
strand DNA seems to be correctly copyed, as i can see an shift
upwards in the experimental (against the control) by DNA migration
in an agarose gel.  I used either T4 (plus and minus gene 32 protein)
and then thinking because I had 5.2 kB (insert + pGEX vector),
used T7 DNA polymerase for the polymerization reaction. I also
tried different bacterial strains, such as TG1, DH5a and W3110,
instead of the original MV1190.  In all cases, using from 10-1000
ng of DNA estimated from a gel , i got either no colonies or few colonies withou
t any presence of the mutagenized DNA
(the transformation is working properly with a control
DNA). Is there someone who have experience with this kind of
trouble and can try to help me?

Thanks in advance ,

Desperately seeking mutants


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