PCR Woes.

Mr. John P Phelan jphelan at hgmp.mrc.ac.uk
Tue Aug 14 10:38:35 EST 2001


Dear All,
 
Apologies for my lack of info in my previous e mail. Here's the specifics. 

I am trying to PCR up a 3.5 kb fragment from a plasmid using pfu.

Basically I am seeing alot of smearing at the
top of my gel, most probably template.  There is unspecific banding
(2.5kb) in some lanes and I can see my primers at the bottom of the
gel. My target product is approx 3.5kb. I have messed around with lots of
parameters such as increasing and decreasing annealing temp but to no
avail.

Also, I am using pfu (Stratagene) which is a high fidelity polymerase so
my extension
times are quite high, i.e. 8-10 minutes. I have tried 15 cycles in some
cases and in others 30. Both cycle times should work as there is plenty of
template available but nothing. 

Someone also suggested to me reducing the extension temp by 4 degrees to
68 but so far nothing.

I am confident about my Taq and I am also going to re-aliquot fresh
primers as I have been freeze-thaawing my current ones for a few weeks
now. I buy Roche dNTPs (already aliquoted) so I dont think they are at the
problem.I am also using fewsh x10 buffer. 

Basically I was wondering if there is something fundamental I should know
about attempting to PCR long products. 

If anyone can input any ideas I would be extremely grateful. 

Best wishes.

John

*****************************************
John P Phelan
Bloomsbury Centre For Structural Biology, 
School of Crystallography,
Birkbeck College,
University of London, 
Malet Street, 
London.
WC1E 7HX

E mail jphelan at hgmp.mrc.ac.uk

0207 631 6807 (Office)
0207 631 6868 (Lab)
0207 631 6803 (Facsimilie)
**************************************** 





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