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Problems with QuickChange mutagenesis kit

Problems with QuickChange mutagenesis kit - Protocols and Methods Forum

Problems with QuickChange mutagenesis kit - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-09-2006, 10:25 AM
María Jesús García
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Default Problems with QuickChange mutagenesis kit



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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  #2  
Old 06-09-2006, 05:45 PM
ChenHA
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Default Problems with QuickChange mutagenesis kit

On Fri, 9 Jun 2006 12:25:12 +0200, María Jesús García
<[Only registered users see links. ].es> wrote:


If it doesn't work at all with anything, then one problem might be the
polymerase. I find that if you put the pfu polymerase into -80 deg
freezer, it won't work well anymore.

Assuming that your enzyme is still working, other things you can do -
lower the annealing temperature, use longer extension temperature.

You can also try other polymerase like KOD which other people have
used and found to work. It is supposed to work faster than pfu, but
I haven't tried it for Quikchange so I wouldn't really know.




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