72C or 68C extension temp.
Dr. Duncan Clark
duncan at nospam.demon.co.uk
Thu Oct 8 09:06:44 EST 1998
In article <361860bc.412809 at news.uni-hamburg.de>, Soenke Behrends
<behrends at plexus.uke.uni-hamburg.de> writes
>when you buy an enzyme mix for long PCR like expand from boehringer
>the manufacturer tells you to use 68C extension temperature when the
>pcr product is above 3 kb and 72C if it is below. I am currently
>trying to amplify a 3 kb pcr product. What is the rationale behind
>the choices for the extension temperature and what about using
>for example 70C. Any theoretical thought or practical experience
>Thanks for your time.
>behrends at plexus.uke.uni-hamburg.de
It all dates from one of the original Wayne Barnes's papers on his
invention of long PCR. Basically he found that unless he dropped the
extension temperature from 72 to 68 he couldn't get reliable long PCR or
at times even any long PCR products. Why? Who knows?
Having said that I wouldn't call 3kb long PCR unless it is a human
genomic product. I define long PCR as above 2kb on human xsomal and 10kb
on lambda i.e Taq pol on it's own will have severe problems amplifying
these efficiently whereas a mix will easily cope with these and beyond.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
More information about the Methods