help me dilute this - (Nov/08/2010 )

I have 6 primers at 100uM: Primer 1 in forward direction, Primers 2-6 are in reverse direction. I have to make a mix so they are all 20uM in the mix. I would like the mix volume to be 50ul, but not sure if that's possible.

If I take 10uL of the first 5 primers, I will have 20uM per primer at 50uL final volume. But I have 6 primers here. What's the easiest way to do this?

10uL of primer for a 50uL final volume at 20uM.

you seriously want to mix 6 different primers for 1 reaction?
either you should have increased the initial concentration... or increase the final volume... or check the protocol again?

vetticus, yes multiple primers can be mixed together in certain experiments, and is actually required in order for the experiment to proceed.

OK, forget the 6 primer mix for now.

Instead how would I mix 16 primers together from a 100uM stock to have each primer be 20uM final in the mix? I have figured out so far that I can make sets of mixes of 4 that are x uM (set1, set2, set3, set4). Then mix each of the 4 sets into a 1:1 ratio so that the final concentration of each mixed set is 20uM final hence all the primers in that mix are 20uM.

My question now is, say that I mix 2ul of 50uM of set1 primers with 2ul of 50uM of set2 primers. Will this 4ul mix contain 50uM of each primer set or 25uM?

claritylight on Tue Nov 9 01:09:31 2010 said:

Clarity,

Thanks for the mental jog, but unfortunately, I have not been able to mix the primers in the concentration you require and the volume as well. The multiplex I carry out, I have my fourteen primers aliquoted and I add each primer separately to the final reaction mixture.

As far as your today's question is concerned, the final reaction would have 25uM of each primer (since it is being diluted in a equal volume, thus reducing the concentration to half).

Clarity, fogive me, I should have written, "you seriously want to mix those 6 primers at their concentration for that reaction?"

Given the initial concentrations of the primer stock you have, and the final volume and concentration you want... you can't do it.

So either, order your primers at a higher concentration, or reduce the final concentration.

and gt_ameya answered the other question.

Ok, if I had 5 primers at 100uM stock, would it be correct to mix them 10uL each into a final volume of 50uL, so that they are each at 20uM final in the mix?

bingo!!!