Quick Change

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Charan
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Quick Change

Post by Charan » Mon Sep 26, 2005 1:48 am

Hi,
I am having issues with Quick change mutagenesis. :( :(
I am trying to intrduce a single base change in a gene cloned into a plasmid (5Kb). My oligos are 37 bp long with 18 bp homology on either side.
I am following the protocol as per the manual but only variation is that
a) My oligos are not PAGE purified
b) I donot use the super competent cells for transformation. I make my own(chemically competent).
At the end of it I get NO colonies.:shock: :shock: :shock:
Is my reaction not working or my transformation?????????/
In either case what would you recomend???????/
PL. chip in with idea(s). Any suggession(s) wil be appreciated.

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canalon
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Post by canalon » Mon Sep 26, 2005 2:09 pm

At least you should have a transformation control, is it working? If not The problem is coming from your cells, if it does, from your mutagenesis, but I am not familiar with thi sprotocol, so I can't help...

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