Kunkel mutagenesis- Help needed

Jonas Lindeberg Jonas.Lindeberg at bmc.uu.se
Fri Oct 7 11:13:57 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

You have the possibility to use PCR-mutagenesis with the primer that you
already have. You will need another primer for this though. The basic
strategy is to make two parts of your gene product with PCR and ligate them
with a second PCR-step. This means that you also will need PCR-primers at
the ends of your entire PCR product. You can also use the primers in your
vector. This strategy works well for me. If you are still interested to
learn more details about this let me know.

Best of luck! Klas

E-mail: Klas at bmc.uu.se






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