Site-specific nucleotide base mutagenesis 2

drew smith drsmith at silver.ucs.indiana.edu
Tue Jan 9 16:12:40 EST 1990


In article <9001092026.AA25497 at genbank.bio.net> J_MCLAUGHLIN at hvrford.bitnet writes:
>I run 1% agarose gels of the
>DNA both before and after copying and it has indicated copying did occur.  I
>then transfected into E. coli strain XL1Blue dut+ung+.  I get lots of plaques
>but no mutations so lets here what everyone thinks.

The important control is the mock reaction with no primer:
you could be getting priming from small RNA contaminating
your DNA prep.  This is one way you can get lots of DNA
synthesis, but no mutants.

You might also want to try a polymerase other than T4.  I've
always found its lack of processivity to be a problem, and I
don't know how much the gene 33 protein will help.  Another
approach to the processivity problem would be to clone into
a smaller phagemid vector.

Finally, sometimes you just happen to hit a repair hot spot.
If all else fails, clone your gene backward and try again.
- Drew Smith
  Dept. of Biology, Indiana University



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