Kunkel mutagenesis- Help needed

LEMAILLET lemaille at msdos.montpellier.inra.fr
Thu Oct 6 08:56:20 EST 1994


Dear netters,

I want to perform a single base mutagenesis using the Kunkel method, and I can 
only check it by sequencing. First, I produced single stranded DNA containing 
my target sequence in CJ236 with no uridine in the media. I made the second 
strand synthesis and ligation using T4 polymerase & ligase, checked the 
displacement from single stranded DNA to double stranded on gel and transformed 
XL1Blue with that product. I sequenced as many as twelve clones and obtained no 
mutation.
So I did the same thing but added uridine in the CJ236 growth media. I obtained 
phage particles which titer was four times higher in CJ236 than in XL1Blue. I 
sequenced 6 clones this time and, once again, obtained no mutants.
Here come my questions:

- Do you think that the titer of the phage is high enough ? If not, how could I 
increase it?
- Do I have to screen more mutants ?
- Is XL1blue suitable for this method ?
- My primer is 18 base long with one mismatch in the middle, Is that enough?
- Could I use this primer in another method, more efficient ?

Sorry for this long explanation, but I really want to understand why it doesn't 
work!
Any help greatly appreciated.Thanks in advance for your time.

Guy LEMAILLET
ENSAM-INRA-CNRS
MONTPELLIER
FRANCE

e-mail: lemaillet at msdos.montpellier.inra.fr





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