Long PCR

Bryan L. Ford fordb at bcc.orst.edu
Wed Oct 1 14:16:05 EST 1997


Rajesh Kumar wrote:
> 
> Hi,
> 
> I need to do a long PCR (~9 kb). Can somebody suggest me a suitable
> polymerase and a source of it ? How about using a mixture of two
> polymerases, say, Taq and Pfu ?
> 

Rajesh:

Early in the long PCR era we used our own combinations of enzymes with
success. However the performance of Boehringer-Mannheim Biochemicals
"Expand" long PCR system was noticeably better than our own mixes so
that we now use theirs exclusively. Generally we follow their
instructions closely except that we typically use half the enzyme
concentration since the polymerase is a bit expensive. The maximum
product length I have needed to do is 9.6 kb. I have found that keeping
your template intact is an often unmentioned variable in getting
successful long products. So for example be careful to avoid 
unnecessary freeze/thaw cycles-- and if they are necessary use either a
minus 80 degree ethanol bath or LN2 to "snap" freeze before storage at
-20 C.

Good Luck,
Bryan




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