Long PCR

Mark S. Rose msr2 at cornell.edu
Thu Feb 25 20:56:13 EST 1993


From: Miguel Zamora, miza at hardy.u.washington.edu

>I'm having problems amplifying a 5.3 kb DNA fragmente using PCR. I've
tried
>Taq Polymerase and Hot Tub (Amersham), and I didn't get anything. I know
>the reagents are OK because I get the right size product if I use two
>primers that are closer to each other. I've amplified some large
fragments
>before (up to 2.6 kb) with no problem. Anyone has any experience in long
>PCR amplifications. Any suggestions? 


When amplifying large products it is important that you provide an
extension time of sufficient length to allow the synthesis of large
products.  For a 5.3 kb product I would allow at least a 4 minute
extension at 72 degrees. You might also try increasing the amount of
dNTPs in your reactions since they may become limiting in the synthesis
of large PCR products.  Additional amplification cycles might also prove
productive.  If at first you don't succeed...........
Good Luck 
mark
msr2 at cornell.edu
Dept. of Plant Pathology, Cornell U.
Ithaca, NY, USA



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