In article <854842535.30333 at dejanews.com>, u8251012 at cc.kmc.edu.tw wrote:
> Dear netter:
> I have Some questions about PCR (polymerase chain rex)
> and need your advices !
>> 1.How can I remove (or reduce) the primer dimer in PCR reaction
> how about increase annealing Temp ? or lowering Ma2+ conc.
Work to optimize PCR with primers that form dimers is usually not worth
trying. Order new primers that are designed more carefully (often adding
or deleting one base at the 3´end is sufficient). It is sometimes not
obvious that a primer pair forms dimers, so I prefer to run it through a
PCR computer program (e.g. Amplify) before ordering.
> 2.After 38 cycles, a faint signal was obtained. Can I increase
> cycle number, or template DNA or even dNTP content
> TO further INCREASE THE SIGNAL I WANT ??
Using more that 35 cycles is usually not needed for amplification of DNA
fragments as long as you make sure that (1) primers fit the template
optimally (2) annealing temperature is ok for the primer pair in use (3)
no primer dimers are formed.
Point 3 might be the reason for your particular problem. -> see above
Other points to increase PCR yields: (1) optimize conditions by titrating
magnesium (2) add pyrophosphatase (3) add goodies such as 10% DMSO or 3%
Triton X-100 (final concentrations, no typo!). Not recommended is (1)
adding excess Mg++ or dNTP, as this might decrease specificity and
definitely increases misincorporations when you use Taq polymerase (2)
overcycling (3) reamplification if you use Taq (not so much of a problem
with proofreading polymerases)
> 3.What's the best methods to elute out the PCR product with
> high yields and low DNase contaminated. ANY kits suggested ?
DNase contamination is never a problem in PCR (at least has never been to
me): all DNases that might have contaminated your sample are inactivated
by PCR. If you have a problem with contaminating DNases, always keep your
samples frozen in TE until use, and boil your DNA samples for 10min BEFORE
adding PCR buffer (that contains Mg++).
For purification I routinely use the NucleotraPCR kit from Machery-Nagel,
Germany, or gel purification with low melting point agarose and agarase
digestion (the latter is good for PCR that gives different products). You
can try other kits (e.g. Qiagen) as well, if they work relyably for you
(they don´t in my hands).
Hope this helps,
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz