PCR Woes.

[Mr. John P Phelan]

Hi there,
. I am trying
to PCR up a 3.5kb insert from a plasmid and its not working. Are there any
fundamental changes to parameters I need to implement in order for it to
work. I am
using pfu with extension times of up to 8 minutes and longer. I have tried
it at 52, 54, and 58 degrees. I also decreased my extension temp to 68 to
see if that would work, but no.

Can you help?
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)
****************************************