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

Roger "Don't call me Aman" Morton sbyarm at rulsfb.LeidenUniv.nl
Tue Jul 19 06:33:50 EST 1994


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.*
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---------------------------
Roger Morton
sbyarm at rulsfb.leidenuniv.nl



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