deletion mutants

[Wolfgang Schechinger]

Dear Ajith, 

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!

Wolfgang


> 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 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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usual disclaimers apply 
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