4 replies to this topic

[cjmills]

Hi everyone,

I am trying to optimize a multiplex PCR and am missing a band. I want to try to run my control with just 1 pair of primers and am confused as to how to calculate the volume required. My rxn volume is 25 ul, the final concentration of each primer is listed as 6.25 pmol. The initial concentration of my primers is 12.5 uM. My "go to" equation is (C1)(V1) = (C2)(V2), but I don't know how to used this when C1 is in uM and C2 is in pmole (not pM). Can anyone help me? This is driving me crazy. Thank you!

[Trof]

You have to make clear if you need a concentration - uM, pM (means umol/L, pmol/L) or amount (umol, pmol only).

If there is supposed to be 6.25 pmol in 25 ul reaction, that means final concentration is 6.25/25 = 0.25 pmol/ul = 0.25 umol/L = 0.25 uM.

You can then use the equation, I think.

I count it differently. You need 6.25 pmols in reaction (which is by the way not much, I usualy use 10 pmols), in 1 ul of your stock solution there is 12.5 pmols (12.5 uM = 12.5 umol/L = 12.5 pmol/ul) so:

1 ul .......12.5
x ul ........6.25
x = 0.5 ul

So you need 0.5 ul of each primer

[cjmills]

Thank you so much for your reply, Trof. I thought there were 10^6 pmole in 1 umole? Shouldn't the final concentration be 0.0025 uM?

That is what I keep getting and it seems like a ridiculously low number.

Thanks,
Carrie

[Trof]

cjmills, on Jun 16 2010, 06:17 PM, said:

Thank you so much for your reply, Trof. I thought there were 10^6 pmole in 1 umole? Shouldn't the final concentration be 0.0025 uM?

There is,  but there's 10^6 ul in liter. So pmol/ul equals umol/L.

[cjmills]

Duh! Thanks Trof! I could not figure out what I was doing wrong.