Concentration specification in PCR - (Feb/06/2014 )
I've a paper that gives me the following ingredients of a PCR in a 25 microlitre volume:
10 pM of each primer
0.2 mM dNTPs
2 microlitre DNA
1 U Taq
My question is about the concentration of the primers, since 10 pM (which I read as 10 pmole per litre) would be much too little...or have I a wrong approach?
Thanks for any input.
It is not pM but rather pmol, which is entirely different. In a 25 ul reaction, you would need 1 ul of a 10 uM oligo (10^-6 liters x 10^-5 mol/liter = 10^-11 mol).
Thanks, therefore it's an amount not a concentration...anyway your calculation I didn't get.
I'd calculate: 25 uL reaction contain 10 pm primer (final amount), 1 uL of the primer solution (10 uM = 10 pmol/uL) contain 10 pmol, so I have to add 1 uL to the reaction.
(and I start wondering if the 0.2 mM dNTPs are meant as concentration or amount.)
I'd strongly suggest you never write pmol as pM (or as pm), which will lead to just the confusion you see.
The protocol writer could have specified the amount of primer needed as 400 nM (a 25x dilution of the 10 uM stock).