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Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Jul 29 16:26:21 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
    I agree that using PCR to do mutagenesis can be troublesome, unless you
wnat lots of random mutations, and I strongly suggest looking into the
Altered Sites kit from Promega. This system couples selection for the
mutants you want with selection for AMP resistance, so screening is a breeze
(mutant yields are about 90%). The vector they supply has a mutant bla gene,
and is AMP sensitive. During the mutagenesis step, you use a second oligo to
repair the bla defect, and thus have a selectable marker for plasmids which
have been mutated. This system has been used in our lab for several months
now, and it almost always works the first time. One person in our group has
made mutations in three consecutive amino acids using this system. Good luck.

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