PCR re-amplification

Stephen Klautky agoodrid at vaxa.weeg.uiowa.edu
Wed Jun 3 18:13:00 EST 1992


In article <1992Jun2.053705.14020 at talon.ucs.orst.edu>
listona at bionette.cgrb.orst.edu (Aaron Liston) writes:

>I would like to hear from anyone who is successfully re-amplifying
>PCR products.  How do you do it?

In our lab success seems to vary from template to template.  Most
of the DNA we have worked with reamplify readily using the same
conditions and primers as in the first amplification.  I should
add that we have been using BMB Taq for most of our experiments
(we have also used Promega Taq).

The method you use to isolate the fragment may affect the ability
to reamplify.  So far, I have just diluted the product from the
first rxn 1:100 for the second amplification.

Some in our lab have had problems with certain fragments but they
seem to be the exception.  People I have talked to in other labs
nearby don't seem to have any problems in reamplifying a PCR
product.

One of our postdocs had trouble with a 400 bp fragment and eventually
synthesized two nested primers to get a product.  The first PCR
product was made from a 1st strand cDNA mix.  Usually, we are able
to use the original template so that reamplification is not
necessary.

I don't know if anyone has been keeping tabs on the pitfalls of
reamplification.  We set the parameters for the first PCR rxn so
that a full length product will predominate.  In many protocols there
is a post-PCR extension step -> 15 min at 72 deg C.  Hopefully this
ensures that incompletely synthesized products are completed at
the end of this step.

I have not tried to leave out the post-PCR extension step to study its
effect on reamplification.  So essentially, we add it because it
sounds like it would be appropriate.

You might also consider the design of your 1st PCR amplification.  If
you are doing many cycles (35-40) then some of the polymerase may be
inactivated by the last few cycles (depending on your cycle parameters).
A low amount of polymerase could leave the ends of your products ragged
and therefore unamplifiable in the second round with the same primers.
The advertisements for Vent and Pfu polymerases claim that those enzymes
are more thermostable than Taq.  Their use may eliminate this potential
problem.

Hope this helps.


-=] Steve K. [=-          AGOODRID at VAXA.WEEG.UIOWA.EDU

My opinions are my own blah, blah, blah.
(P.S. Call Cetus tech services (if you have not already) they may have
some pointers for you.)



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