primer dimers and the 2.4kb PCR product that used to work?

szcooley at chip.ucdavis.edu szcooley at chip.ucdavis.edu
Wed Jul 20 11:47:30 EST 1994


Roger "Don't call me Aman" Morton (sbyarm at rulsfb.LeidenUniv.nl) wrote:
: Dear Satisfied PCR customer,
: My PCR used to work. Now it has stopped. Can't seem to work out why.
: The system. 2.4kb product. 21mer primers. Used to be able to amplify
: this product from plant DNA transformed with the plasmid. Now I can't.
: The primers produce a product with 1ng of plasmid but not 10pg.
: Each primer works OK with a different primer nearby using plant DNA 
: (ie primers are OK to produce a short product).
: When trying to amplify the long product from plant DNA I get a primer 
: dimer sized product. But I also used to get this when the amplfication 
: was working. However, I think the primer dimer is related to the failure
: of the reactions because it is fainter in reactions that work than ones 
: that don't. 
: The primer dimer appears in water only reactions too.
: I am limited in my choice of primers so it is difficult to move them.
: Any suggestions gratefully read.
: yours
: *************************************************************************
: *I do PCR and I am as mad as hell and I am not going to take it anymore.*
: *************************************************************************
: ---------------------------
: Roger Morton
: sbyarm at rulsfb.leidenuniv.nl

The production of primer diamers naturally competes with the "real" 
product, so your observations make sense. I also have had cases where a 
set of primers worked for a while and then failed (at least in situations 
where the best efficiency is needed). Resynthesizing new primers fixed 
the problem. Operon (a company in California) claim that primers go bad 
at -20 oC. Currently I am storing mine at -70 oC or lypholizing aliquots.

Mike Cooley      mbcooley at ucdavis.ucdavis.edu



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