Very low PCR primer concentrations? - (Feb/15/2007 )
I have inherited a PCR protocol for a 700bp amplicon from a colleague who is no longer in the lab. In the PCR recipe she published recently in a paper, she said she used 14 pmol primer/20 uL reaction. To me this seems a very small amount - I usually use 0.1 to 1 uM primer. However I have dutifully followed her recipe and didn't get any bands.
Is this amount of primer far too small or should I trust what she has published and keep plugging away at it, changing template etc. to see if this is the problem?
I have used 20pmol primer in a 20 ul reactionand always worked.
Check the conditions for the enzyme that you are using for amplification.
rach_b
14 pmol is an absolute amount, but 0.1-1 uM is a concentration. If you have a primer concentration of 1 uM in a 20 ul PCR reaction, you have 20 pmol of primer in that 20 ul. If you set up a PCR with a 14 pM concentration of primer, as you may have done, the absolute amount of primer in there is only 280 (14 x 20) attomol, which is not much, and your reactions will run out of primer very quickly.
Hai
0.1 micmolar seems just suffieicnt for your reaciton. check other conditions
good luck
madhan
If it helps: pmol/µl = µM; that means in your case 14pmol/20µl=0.7µM
good luck!
14 pmol is an absolute amount, but 0.1-1 uM is a concentration. If you have a primer concentration of 1 uM in a 20 ul PCR reaction, you have 20 pmol of primer in that 20 ul. If you set up a PCR with a 14 pM concentration of primer, as you may have done, the absolute amount of primer in there is only 280 (14 x 20) attomol, which is not much, and your reactions will run out of primer very quickly.