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-   -   please help me-basic information about site-directed mutagenesis PCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/3323-please-help-me-basic-information-about-site-directed-mutagenesis-pcr.html)

trypsin 05-01-2008 05:14 PM

please help me-basic information about site-directed mutagenesis PCR
 
I have to change a codon in my plasmid vector. It should be changed by site-directed mutagenesis.
Now I know that i made any mistakes. I have no idea which kind of parameters and substances are important to get a good result.

Somebody told me I must use a primer which has minimum 30-35 bp and a GC-end. She told me the annealing temperature should always be 55C independent of the primer length (what i don't understand). I have two protocols: the first describes the use of only one primer (forward primer with the changed na) the second describes the use of two primers (forward and reverse primer). What is the right or better protocol?
I use the pfu high fidelity polymerase at 68C. Is this the right/optimal temperature? Or should i use another polymerase? How many cycles should be do? I know that 35 cycles are too much.
I would be very happy if someone could get me some information or tips.
thank you.
trypsin

kmunson779 05-01-2008 06:21 PM

Re: please help me-basic information about site-directed mutagenesis PCR
 
I follow the guidelines in the Stratagene QuikChange [Only registered and activated users can see links. Click Here To Register...], except I usually put my mutation at the 5' ends of my primers rather than in the middle. Stratagene also has a free [Only registered and activated users can see links. Click Here To Register...] to design primers.


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