Help Priming with Low Tm primers
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!
how did you design your primers? What are you trying to amplify (e.g.
eukaryotic, prokaryotic, animal, mineral, vegetable....)? What is your
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>
"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
-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
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