Real Time PCR dilution

[prathap]

Hello,

I am new to Real time PCR work. My custom synthesized primer stock conc is 10nM (nanomoles) and would like to make my working stock conc of 300nMolar (nanomolar). can some one help me out to make my working conc.

Thanks in advance

[admin]

This is an easy calculation if you use the formula

C1*V1 = C2*V2

where C is concentration in nM
and V is volume in microlitres or nanolitres

Also see this:

PCR Primer Dilution

[prathap]

thanks for message.
My primers for real time PCR stock conc/Amount is 10nmol and I want make working conc for 300nM. I guess C1V1=C2V2 will not work here because my stock conc is in nMOl and working should be in nanoMolar

My probe stock conc is 100 pmol/microlitre and want to make working conc for 100nM. please guide me in this regard

thanks in advance

[admin]

Hi you can still do this by using a primer molecular weight calculator, using your sequence (input GCAT sequence) then you can calculate your needed concentration from the moles and calculated molecular weight.

[namishu95]

I am confused about your working stock conc that primer was at 300nmol and probe at 100nmol, most working stock is from 5µm/µl to 40µm/ul, did you mean the final primer/probe reaction conc( add 0.5ul primer or probe to 25ul final reaction), if so, you should make primer working conc as 15µm/ul and probe as 5µm/ul,
add 100µl water to your original stock conc primer (10nmol) to gain 100µmol Further Stock Conc (FSC) primer, then dilute 15µl FSC primer into 85µl water to gain 15µm/ul.
dilute your stock conc probe as 20 times to gain 5µm/ul working conc probe.