Problem with PCR of 2 kb linearised plasmid.
vmiao at oregon.uoregon.edu
Wed Aug 18 21:09:57 EST 1993
In article <1993Aug18.112550.204815 at uctvax.uct.ac.za>,
mybdav02 at uctvax.uct.ac.za wrote:
> 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.
> 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).
1. Desperation is not the solution. (Maybe later).
2. Would using a longer extension time (e.g. 2 min) increase the yield of
the correct fragment? A general rule is 1 min/kb.
3. Although your oligo is 25 bases long, only 14 of it is homologous to
the template in the first few rounds of amplification; is that taken into
account in your Tm calculation? Maybe you could lower the annealing
temperature for the entire program? If low Tm of the primer is indeed the
problem, then perhaps you could use a lower temperature for the first few
cycles to encourage the "right" reactions, and then increase it in later
cycles to favor continued production of the correct band (after a few
cycles, the right reactions will have 25 bases of homology) and discourage
the "wrong" reactions (e.g. did 65 degrees reduce the amounts of the
Even if you don't manage to shake the spurious bands, maybe you could
increase the yield of the right band enough to gel purify and use for your
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