Help Priming with Low Tm primers

Ted M. tedm at darkwing.uoregon.edu
Wed Aug 23 22:04:52 EST 1995


In article <1995Aug22.193056.22313 at emba.uvm.edu>, rfialho at moose.uvm.edu
(Rob Fialho) wrote:

> I designed these 18 mer primers to amplify a 700 or so bp fragment but 
> they dont seem to work under any condidtions, I get multiple bands 
> anywhere between 48-50 C. Their Tm's are 50 and 54 C.  I have lowered the 
> magnesium concentration to 1 mM and still got faint multiple bands.  How 
> can I get around this?  I'd aprreciate any suggestions!  Thanks!
> 
> Rob



Rob,
A simple Hot start may help the specificity, try heating the mix sans Taq
to ~80°C and then add the Taq, go directly to the initial denature temp
and cycle as before. Possibly pull a sample at 10 cycles to see if your
specific product is being washed out by nonspecific products in later
cycles. Higher anneal temps is the ticket to specific priming but a hot
start can make all the difference.
good luck!

regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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