Problem with PCR of 2 kb linearised plasmid.

[mybdav02 at uctvax.uct.ac.za]

Hi folks

I'm having some problems trying to PCR up a 2 kb plasmid (pWM521).  I have
linearised the plasmid with SmaI and then designed primers for the ends
that have been created.  The primers are 25 bases long, and have an 11
base overhang (the overhang contains unique restriction sites).  Their Tm's
are around 67 deg.

I user the following conditions for my PCR:

   92 deg  2 min
   92 deg  45 s )
   60 deg  60 s ) cycle 30x
   72 deg  90 s )
   72 deg  5 min

  0.2 uM primers
  30 ng  template
  1.5 mM MgCl2
  0.2 mM dNTPs
  Promega 10x Taq buffer OR special buffer containing 300 mM Tricine
                                                      0.1 % gelatin
                                                      1 % thesit
                                                      50 mM mercaptoethanol
  2.5 U Taq
  Water to 50 ul
  50 ul paraffin to cover solution

  Using these conditions, I get three bands on a 1% agarose gel.  The top 
band is the correct length, and the lower two correspond to unrelated lengths.
I have tried increasing the annealing temp to 65 deg to reduce non-specific
binding of the primers, but this results in a decrease of the desired product.

Is there something that I am doing wrong??  I've checked all my calculations
many times, as have various people in my lab.  I have a bad feeling that the
bands are due to binding elsewhere in the plasmid that is tighter than at the
desired position.  

If anyone has any ideas, please let me know - I'm getting fairly desperate as
I have to get my insert cloned by the end of September (the insert has already
been amplified by PCR - its conditions also don't work for pWM521).

Thanks in advance

Dave

Dave Myburgh
Dept of Biochemistry
University of Cape Town
mybdav02 at uctvax.uct.ac.za