Help Priming with Low Tm primers

Hans Beernink hbeernin at med.uvm.edu
Wed Aug 23 08:24:55 EST 1995


Rob Fialho (rfialho at moose.uvm.edu) 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! 

Hi Rob,

how did you design your primers?  What are you trying to amplify (e.g. 
eukaryotic, prokaryotic, animal, mineral, vegetable....)?  What is your 
template source?

Basically, PCR will amplify anything that is primed.  Thus, if your
primers stick anywhere else, it will amplify.  This sounds pretty obvious,
but lots of people forget that certain regions of DNA are repeated.  If 
your primer is specific to one of these regions, you may not get a single 
band- no amount of fiddling will give your desired product if the primers 
can anneal to multiple sites that are identical.  Now, if you are 
positive that there is only one specific site, you need to optimize 
according to temperature _and_ Mg.  Additionally, look at the primer 
sequence to make sure that they cannot pair to eachother.  Finally, there 
are some tricks to programming-  try a high annealing temperature for the 
first 10 cycles or so (around 55C, but you may have to play with it), 
then lower the temp to 45-50.  This allows for highly stringent annealing 
initially (low efficiency of amplification, but highly specific).  

If none of these work, post some more information on your specific
experiment, or email to <hbeernin at protein.med.uvm.edu> or 
<hbeernin at moose.uvm.edu>

Best regards, 

Hans
--
_____________________________________________________________________________

"The worst monotonous drone coming from a lectern or the most eye-splitting
textbook written in turgid English is nothing in comparison to the 
psychological Sahara that starts right in your bedroom and spurns the 
horizon."

	-Joseph Brodsky, from "In praise of Boredom"
	 delivered as a commencement address at Dartmouth College.
_____________________________________________________________________________


Hans T.H. Beernink, Department of Biochemistry, University of Vermont
hbeernin at protein.med.uvm.edu 



More information about the Methods mailing list