Soeing PCR- not getting the mutation- even tho its in the primer
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
>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.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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