Annealing adapters

[IdahoRAD]

Hello all--

I work on preparing RAD libraries, and have a question regarding annealing adapters.

Suppose my adapters' top and bottom oligos are each at a stock concentration of 100 uM. If I pool each adapter equi-molarly prior to annealing, will I have 100 uM of annealed adapter or will I have 200 uM of annealed adapter?

Of course, I am simplifying my question, because the thing that is confusing me is the molarity-result of pooling equi-molarly. I have searched for an answer, to no avail! I know this is a basic question, but it has really been stumping me!

Thank you,

Cody

[nucacidhunter]

You will need concentrated annealing buffer to dilute your oligos for maximum annealing. In the simplest form if you just mix equal volume of 100 uM oligos you will get 50 uM adapters.

[IdahoRAD]

Nucacidhunter--

Thank you for your reply. Would you mind explaining your rationale, in the example you gave above? Is it because if we pool equi-molarly, we are doubling the volume and thus the molarity decreases?

Suppose I pooled 100 umol of each component of the adapter, will I thus have 100 umol of annealed adapter?

The person who was in charge of adapter preparations at my institution left rather abruptly, so I am trying to figure this out on my own. Seems like with these simple protocols, we (at UIdaho) take them for granted until we have to do them ourselves!

[nucacidhunter]

If X mole of oligo A and B is annealed (the same molar concentration) it will give X mole of adapter. In the process the volume has increased by 2x, so the concentration will decrease by 1/2x.

[kerplunk412]

In case you are looking for second opinions, I completely agree with nucacidhunter.