manta

Hi guys,

Is there any reason why I could not use a real-time machine (standard AB7500) to run conventional PCR? We have only one regular thermocycler in the lab, and it´s currently in use. I figure the steps are the same, but maybe I´m missing something. I wouldn´t add any probes to the reaction, and of course would discard whatever wacky amplification plot I would get. It´s the first step of a nested PCR (hopefully the other group will be done before I get to the second step)...I´ll eventually run it on a gel.

Thanks!!

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!