Hi there,

I'm rather ashamed to ask this question but need re-assurance and advice for making dilutions of my primers for a 5'RACE PCR. I am trying to amplify heavy and light chain antibody sequences from cDNA.

For the PCR reaction, I need to run 200 nM of either heavy or light chain primers. My primer stocks are 100 uM, and so normally I dilute them to 10 uM and use 1 ul in the PCR reaction.

For the heavy chain primers, there are two of them and they need to be equimolar, so I make stocks of each at 20 uM, and make a 1:1 mix so the final conc. is 10 uM, allowing me to add 1 ul in the PCR. No problem there (I think).

The tricky part is with the light chain primers. I have 3 kappa primers, and 3 lambda primers (100 uM each). I've been advised to make equimolar mixes of kappa and lambda respectively, but the final primer mix consists of 90% kappa and 10% lamdba to reflect the known light chain isotypes in my species.

This final 90/10 mix needs to have a final concentration of 10 uM but I'm struggling to work out how to calculate this and I could use some help.

Many thanks in advance.