Wolfgang.Schechinger at med.uni-tuebingen.de
Mon Oct 16 11:55:06 EST 2000
if you already have your cDNA clone it's quite simple. All you need
is some mutagenesis primers and the quick change protocol (from
stratagene's websites or search the archive of this NG).
Once you know the triplet you want to omit, have synthesized two
complementary primers flanking the site to be altered for approx 15
bases each and make a PCR of 25 to 30 cycles using Pwo or Pfu
polymerase (extension tim at least 1 min per 1000bp) on approx 0.5 ug
of template, then digest the parent DNA with DpnI, transform the mix
into E. coli and selcet or sequence for mutants.
If you should happen to have flanking primers (e.g. T7 and Sp6
sequenceing primers in many cases), you instead might perform 2
pcrs using 1 flanking and 1 mutagenesis primer, mix the purified
bands and perform a second pcr on them using only the flanking
primers. Then clone the PCR product into the vector again and
selct/sequence for mutants.
Lots of fun!
> From: mathewajith at nccs.res.in ("Ajith")
> Subject: deletion mutants
> Date: 16 Oct 2000 16:46:21 +0100
> Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre
> X-To: <methods at hgmp.mrc.ac.uk>
> To: methods at hgmp.mrc.ac.uk
> hello ,
> i need some information on making deletion mutants . i know the
> complete seq of the gene under consideration , if someone could
> just enlighten me on to go about . thanks .
> Ajith MAthew
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Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de
usual disclaimers apply
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