# Oligo concentration - (Oct/04/2006 )

I know that such questions are posted quite often, but I can't help but asking:

If I have two oligos (which anneal) dissolved in water [1µg/µl] and take one microliter of each and add 18 µl of water or buffer. What is the final concentration of my oligos? What bothers me is the fact that I don't know whether the concentration of the two oligos adds up?? They anneal, so are they considered as one molecule? I'm confused and the funny thing is, our postdoc couldn't help me with this. (And I'm not surprised... )

Well first, you shouldn't really perform annealing based on equimass quantities but rather equimolar. However, if we assume that you get 100% annealing and that the oligos have the same molecular weight then you simply add the two together.

So adding 1 µL of each oligo gives you 2 µg of oligos in 20 µL. This weight of the dsDNA produced will still be 2 µg in 20 µL so that's 0.1 µg per µL.

However, the molar concentration of the dsDNA is only the same the lowest concentration of one of the oligos you use, i.e., 5 µM oligo 1 plus 10 µM oligo 2 can only give a maximum of 5 µM dsDNA.

You should convert to using molar not mass for these calculations.

So adding 1 µL of each oligo gives you 2 µg of oligos in 20 µL. This weight of the dsDNA produced will still be 2 µg in 20 µL so that's 0.1 µg per µL.

However, the molar concentration of the dsDNA is only the same the lowest concentration of one of the oligos you use, i.e., 5 µM oligo 1 plus 10 µM oligo 2 can only give a maximum of 5 µM dsDNA.

You should convert to using molar not mass for these calculations.

Thank you for your fast reply.

I have this protocol by Genscript And they recommend equimass quantities.

But in fact the two oligos have the same molecular weight. So according to your suggestion it should be ok?

These concentration calculations always confuse me.

Yeah, it should be fine (it would work even if they were different it's just you won't get 100%).

However, the mass of the dsDNA produced will be the sum of the masses of the oligos used.