Enhancement of PCR?

[Michael Cooley]

Shahram Mori (smori at nmsu.edu) wrote:
: 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


There were some posts a few weeks ago regarding deterioration of primers. 
Appearently some primers fail to amplify after storage in liquid at 
either 4 oC or -20 oC. It was suggested that the primer form 
intramolecular bonds which limit its effectiveness. The boil/freeze helps 
to restore activity. 


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