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Calculating Primer concentrations for PCR - Is there an easy way to do this...help (Oct/15/2010 )

Hello all,

So primer concentrations:

The primers come in dried form from Sigma and tell you how much water to add to make a 100uM stock solution, I then do a 1 in 10 dilution and make lots of aliquots of 10uM stock. With me so far?

I am using a polymerase for PCR (EXPAND HI-FI) which recommends I use a 3 uM concentration of each of my F and R primers suggesting a volume of 5 ul of each primer (at a concen of 3 uM). So according to the mumber of samples I calculate how much of each primer I will need at a 3 uM concentration (Fine).

My new problem.....I have ordered primers from a different company which tells me how much water to add to the dried primer to make a 100pmol/ul solution. I then dilute this to make a working stock of 10pmol/ul............but here is the dilemma if my protocol for PCR recommends using 3uM concentration of each primer in the reaction how do I calculate how much 10pmol/uL primer stock to use of each of my primers for each reaction.........am so confused.......can anyone help me please??????

I know lots of people have problems calculating primer concentrations - it would be really useful if someone in the know could explain clearly how to go about it. It would certainly make this novice scientist's life easier :)

-phosphate girl-

100pmol/Ál is the same as 100ÁM, so you end up with the same concentrations for your stock and working solutions in both cases (100ÁM = 100Ámol/l = 100pmol/Ál).

-ElHo-

Thankyou for your reply - much appreciated. I feel a bit stupid for not seeing that myself :blink:

-phosphate girl-

Don`t worry about it. It took some time to realize that myself at the beginning of my "lab career".

-ElHo-