Problems with QuickChange mutagenesis kit

María Jesús García mjgarcia at cnb.uam.es
Fri Jun 9 05:25:12 EST 2006


Hi everybody!
I´m looking for some help with the QuickChange mutagenesis kit from Stratagene.
I want to make a single aminoacid change in a 
protein (1.1kb) cloned in pcDNA3.1. (total lenght 
of the construct 6.5kb). I tryed with 50 and 75ng 
of DNA template and 125-150ng of primer. I´ve 
design the primers according to the manufacturer 
and I've used the following conditions for the 
PCR:
Segment 1:
1 cycle 95ºC 30 seconds
Segment  2:
16 cycles 95ºC 30 seconds
55ºC 1 minute
68ºC 13 minutes (2 minutes per Kb)
The problem is that after transformation I DON`T GET ANY COLONIES AT ALL.
I´ve used also the control provided with the kit 
and I don´t get any colonies at all as well.
So, could somebody advise me on where I might be going wrong?
Thank you.

Maria. J. Garcia





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