PCR Amplifying 2-5Kb fragments

Duncan Clark me at privacy.net
Fri Jun 25 03:20:06 EST 2004


Historians believe that in newspost <10dkcsg6kjqrcb9 at corp.supernews.com> 
on Wed, 23 Jun 2004, Phil McClean <mcclean at cableone.net> penned the 
following literary masterpiece:
> Does anyone have a procedure in which they can routinely amplify via PCR
>fragment 2 to 5 kb in length.

Do it all the time from bacterial genomic.

>What are some of the critical factors that
>you have encountered.

It really depends upon the complexity of the genomic source. Human 
xsomal is much more difficult than a bacterial PCR. GC rich is much 
harder than a 50% GC etc.

More enzyme can help i.e. doubling or even quadrupling at times. 4-5kb 
on human genomic is pushing it with standard Taq. Putting in say 2.5u 
Taq with 0.125u Pfu and using the Pfu PCR buffer can make a huge 
difference.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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