Enhancement of PCR?

Shahram Mori smori at nmsu.edu
Mon Mar 6 20:55:30 EST 1995


Richard Hastings (mbxrh at unicorn.ccc.nottingham.ac.uk) wrote:
: I've recently been having some problems with contamination of PCR, i've
: had some advice as to how to minimise contamination and smearing.  One
: such piece of information was to boil the PCR prmers for 5 minutes then
: snap freeze in liquid nitrogen and thaw on ice before using in the PCR
: reaction.  This apparently reduces smearing of products, i found that
: this treatment also enhanced the PCR reaction somewhat, producing
: stronger bands in both my main reaction as well (unfortunately) as my
: no template negative control.
: I generally store aliquoted stocks of primers at -20C then take an
: aliquot and store at 4C and use that rather than refreezing each time
: (which is supposed to have a detrimental effect on the primers).
: Does anybody know why this enhancement is occuring?
: Thanks for any help.

: Richard Hastings (mbxrh at unicorn.nott.ac.uk)
: Dept. of Biochemistry,
: Queens Medical Centre,
: University of Nottingham,
: Nottingham NG7 2UH.

I don't see how freezethawing could affect primers. I have not had a
problem with re-freezing DNA primers. As for Boil/LiqN treatment, I would
THINK that it would allow for complete linearization of your primer (liqN
prevents slow re-foramtion of 2ndary structures). But as to get rid of the
contaminated DNA (which I am assuming is in your primer sample) I don't how
it could help. The best thing to do is to run a PAGE gel of appropriate conc.
and gel purify away your target DNA. Also using Barriertip pipettes will help
future contaminations.
Cheers
 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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