2 replies to this topic

[manta]

Hi everyone,

I just quick validation that I'm not completely messing up my primer dilutions...

I have 2 antisense primers that I need to use in a 1:1 molar ratio, called As1 and As2.

I have 10nmol of each primer in one tube, which I reconstituted in 200uL of buffer. So 10nmol of As1, 10nmol of As2, 200uL buffer = 200uL of 100uM antisense primer mix?

So now, assuming that this stock solution is a 100uM primer mix, I take 10uL of this mixture and mix it with 90uL of water to make a 10uM primer mix for use.

So assuming that this is right so far (please, please let it be right!), this is where it gets more tricky.

My protocol calls for a final concentration of 0.30 uM of the 1:1 antisense mix in the PCR reaction. This is part of a 25uL reaction, so I should be using 0.75uL of the primer mix?

The reaction seems to be working well enough, but I'm having nagging doubts that I am making a false assumption or otherwise skipping a crucial step...but now that I'm writing it out, it looks like it makes sense. Hopefully all is well!

Thanks for your help!

[phage434]

In your first step, you dissolve 10 nmoles of a primer in 200 ul.  This gives a 50 uM solution of that oligo.  Now there is another oligo in the tube, and the solution will also be 50 uM with respect to that oligo.  When they anneal (assuming they do) you still have a 50 uM solution of the pair.  I would call your 200 ul of solution a 50 uM solution.  The rest of your calculation looks correct, but I would interpret the paper as meaning a 1.5 ul addition of the 10:1 diluted stock (i.e., twice as much as you are currently using).  Having said that, there is usually excess primer, and if things are working (which they probably will be) then go with it.

[manta]

Dear Phage,

That makes a lot of sense. I think I'll go ahead and plan to add 1.5ul of the As1/As2 primer mix from now on. The PCR is primarily for viral detection (though we are quantifying as well), so any modifications that can improve the sensitivity, even by a little bit, are worth it.

Thank you for your help!