PCR with 500bp Primer

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Mar 7 11:08:42 EST 1995


Xiaoming Yi writes:

> I have posted this question a couple of days ago, but got not response. Did
anyone do PCR with a big primer like 500bp? If so, can anybody tell me what the
major difference is, what should I do to make it work?

I see two problems:

1) The extra length will stabilize lots of innappropriate annealing at
the stringency you will be constrained to use by the enzyme and the other
primer.  This will dramatically increase your background of extraneous bands,
and may even tie up so much primer that you get no bands.  You can try
to minimize this by keeping the ratio of primer to starting template high,
and the annealing stringency as high as possible by keeping the annealing
temp. high, Mg low, and adding DMSO or formamide.  Note that you'll have
to make sure that the other primer is stable enough to stand up to the
higher stringency also.  Even with all that, I wouldn't bet on this 
working out of a complex template (like genomic DNA).  However,  people
clearly do stuff like this off of simple templates, like plasmids for
in vitro mutagenesis.  If you want to work out of a complex template, I'd
recommend first amplifying with an optimal primer made from just the end
of your 500 n; then reamp with the 500 n primer after the desired product
is formed and specificity is no longer an issue.

2) Keep in mind that on a g/g basis, the molar amount of your 500 n primer
is 20 x lower than the other primer; so you're in danger of inadvertantly
starving the rxn.

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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