Soeing PCR- not getting the mutation- even tho its in the primer

Duncan Clark d_clark at genesys.demon.co.uk
Sat Oct 13 03:56:45 EST 2001


In message <a59bbfb2.0110120801.65df0892 at posting.google.com>, JonnyR9 
<jonnyr9 at hotmail.com> writes on the Fri, 12 Oct 2001
>Dear Beth,
>
>Could you be sequencing the original template?  Whatever contains the
>template material survuves until the end of the reaction, perhaps
>there's more template than product?
>
>eliza at itsa.ucsf.edu (Beth Holleran) wrote in message news:<4a85aa87.0110091515.47b5a949 at posting.google.com>...
>> Hi- I have been doing Soeing PCR for a while but I ran into a new
>> problem- this time I get my two products, SOE them together and when I
>> sequence them they are Wild type! EVEN THOUGH I used primers that had
>> the mutation in them! what in the world has happened? I tried using a
>> lot less template (to reduce the amount of WT template) and everything
>> has been gel purified.  Has anyone seen this? Help! Please!


Cut with Dpn I to kill the original template - Dpn only cuts methylated 
DNA and PCr products are not methylated. Alternatively used alkaline 
denatured DNA as original PCR template. It will not transform.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382




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