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Bernard Heymann bheymann at bragg.bio.purdue.edu
Sat Jul 23 12:26:55 EST 1994

In article <01HF0D7NPA9E000B5I at msvax.mssm.edu>, LAURETTE at MSVAX.MSSM.EDU
(LAURETTE) wrote:

> 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 without 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
> Laurette

    I've just recently gone through six months of failed Kunkel's method
and PCR mutagenesis. I finally switched to the old M13 method and got
mutants almost immediately. It is more work, but it beats having no success
at all! Now, I'm a convert :-)

Bernard Heymann
bheymann at bragg.bio.purdue.edu

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