HOW to Eliminate PCR primer dimer

Mary_Ann_Brow at TWT.COM Mary_Ann_Brow at TWT.COM
Thu Feb 6 16:23:51 EST 1997





The obvious suggestions are to make sure that the primers do not have any
3' end complementarity and to do a "hot start"  (add enzyme or Mg++ at high
temperature).  Beyond that, I have had very good luck with using PCR under
conditions that are similar to asymmetric PCR conditions, but with the
lower concentration primer not low enough to make much single stranded
product.  I usually use 0.5 uM of one primer and 0.05 to 0.1 uM of the
other (e.g., 50 pmoles of one and and 5 to 10 pmole of the other per 100 uL
reaction).  Most PCRs will not yield enough double stranded product to use
up the limited primer, but if this is a concern, that primer can be brought
up to 0.5 uM for the last cycle.






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