Path: utzoo!utgpu!watserv1!watmath!uunet!samsung!sol.ctr.columbia.edu!cica!iuvax!silver!drsmith
From: drsmith@silver.ucs.indiana.edu (drew smith)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Site-specific nucleotide base mutagenesis 2
Summary: controls?
Message-ID: <32691@iuvax.cs.indiana.edu>
Date: 9 Jan 90 21:12:40 GMT
References: <9001092026.AA25497@genbank.bio.net>
Sender: root@iuvax.cs.indiana.edu
Reply-To: drsmith@silver.ucs.indiana.edu (drew smith)
Organization: Indiana University, Bloomington
Lines: 21

In article <9001092026.AA25497@genbank.bio.net> J_MCLAUGHLIN@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