PCR mutagenesis

Andy Kravetz akravetz at Walden.MO.NET
Fri Oct 6 01:28:53 EST 1995


Hi, recently, I posted a way that I was shown how to do mutagenesis via 
PCR which for the references, look in various journals. I am not claiming 
credit for anyone's work. Okay, now that is taken care of, I have a 
problem with a recent clone that I have been trying to make. 

I am trying to change two basepairs and as such, I am using four primers, 
two for each half then I am going to use the halves as template and PCR 
amplify the whole thing. Okay, in the past, it has worked fine but right 
now, I can't get it to work and I need some help. 

the first half worked fine but the second half is not. There are two 
primers, one that is 29mer and one is that 18mer and they are supposed to 
produce a fragment that is 375bp long. But I am getting two fragments at 
475 and 275bp.

Is it possible for PCR fragments to run at different sizes and if so, 
why? Also, I have my annealing temp at 64degrees which I think is 
strigent enough to block nonspecific annealing. Should I go up to 70 
degrees or another temp? 

Thanks all,

andy

--

------------------------------------
"I love the wild power of language and and the purity of the madness that
governs it and makes it music" 
                                        -HST, _Generation of Swine_
Andy Kravetz
Independent Journalist
St. Louis, Missouri
akravetz at walden.mo.net
http://walden.mo.net/~akravetz/



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