simple primer calculation - (Jan/21/2008 )

I seem to have got stuck trying to get my head around a primer calculation- I'm sure it should be simple.

So i've got a stock solution of 10 microM primer. I add 2.5microlitres of this to a 25 microlitre PCR (I know its a strange amount, but its an unbalanced PCR with labelled primers).

So how much, in micromoles, primer do I have in my reaction?

I'll kick myself when i realise how easy this is.....

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Hi there,

so your stock is 10uM=10umol/liter=10 000nmol/1000ml=10 000 000pmol/1000 000ul >=10pmol/ul.

Since you put 2.5ul to your reaction (the volume of your reaction does not play a role in this calculation because you want to know how many primer molecules (=mol) you put), you would have 25pmol in your PCR.

Is that what you wanted to know?

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Yeah thats it. I knew it shouldnt be that hard.
So, just to make sure I've got this nailed.... id I have 0.375 ul of the other primer, thats 3.75pm?

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I've just had a look at a paper by the person I have been collaborating with- when she did her PCRs, she used 0.5uM and 0.1uM of primers- mine seem a lot less, although I am using about the same amount- isnt 25 pm a lot less?

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You said you have: 10 microM primer. I add 2.5microlitres of this to a 25 microlitre PCR, this is a 1:10 dilution so this primer is actually at 1uM
for the second primer you add 0.375 ul, I assume is also at 10uM so in a 25ul reaction this primer is at 0.15uM.

So in fact your primers are at a higher concentration in the PCR reaction than in the paper.

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you have to be careful not to muddle up a concentration indication (eg. 0.5µM) and a substance quantity (25pmol).

25pmol means, if you put that amount into 1l than this solution is 25pM, but you just have 25µl so 25pmol in 25µL is 1µM.

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