site-directed mutagenesis

Dennis J. Templeton djt2 at po.CWRU.Edu
Tue Nov 17 10:11:04 EST 1992


In a previous article, wsun at jeeves.ucsd.edu (Fiberman) says:

>Dear netters,
>
>I am trying to do site-directed mutagenesis using the Bio-rad
>kit.  I am using CJ236 cells and my clone in the Bluescript
>plasmid to make SS DNA with VCM13 helper phage.  Following the
>protocol, I do a negative control where I add everything except
>the mutagenic primer.  However, when I transform XL1-blue cells
>with this negative control DNA, I get colonies on the plate.  In
>addition, when I screened the colonies from my positive plate, I
>don't get any mutants, only wild type.  Can anyone out there
>have had this problem and could offer some
>explanation/solutions?
>
>-fm
> 
>

I think the biggest variable in this reaction (we use the same reagents from
BioRad, since the enzymes in the kit refill are cheaper than by themselves) 
is in the purity of the template SS DNA.  Any contaminating oligonucleotides 
from the prep will prime "randomly" and will result in wild-type background.

Try a couple extra steps of purifying the SS DNA away from any small 
nucleic acids.

Also, double (or triple) check the sequence and orientation of your 
mutagenic oligo. While troubleshooting effots from *other peoples labs* ;=)
we've encountered all too many mistakes of that kind.

good luck,


dennis



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