So my worst fear came true; we have a deadline, we spent the last of the library kit, and our PCR-free libraries all came out well below the minimum for loading to the instrument (MiSeq). The concentrations from qPCR range from 0.14 to 0.7 nM
I remember reading about an alternative denaturing protocol, with HCl used to neutralize the NaOH instead of the large dilution after denaturation. But I did not see any specific instructions about the quantities and concentration of HCl. Does anyone have experience with this?
Edit: I see in the NextSeq protocol they use Tris-HCl pH 7, which enables denaturation of libraries down to 0.5 nM with a final loading concentration of 20 pM. We will have about half of that, so we will probably end up with a rather low cluster density.
Another option is to concentrate the libraries with speedvac, but I am not too eager about this. We tried it before on a library, and we somehow lost everything. Even if we reduce the volume to 1/10th, some samples will still be a bit too dilute.
Another option is to do enrichment PCR, (see my post here, but I am not sure about the primers we have or the conditions.
Any help will be greatly appreciated!
Jon
I remember reading about an alternative denaturing protocol, with HCl used to neutralize the NaOH instead of the large dilution after denaturation. But I did not see any specific instructions about the quantities and concentration of HCl. Does anyone have experience with this?
Edit: I see in the NextSeq protocol they use Tris-HCl pH 7, which enables denaturation of libraries down to 0.5 nM with a final loading concentration of 20 pM. We will have about half of that, so we will probably end up with a rather low cluster density.
Another option is to concentrate the libraries with speedvac, but I am not too eager about this. We tried it before on a library, and we somehow lost everything. Even if we reduce the volume to 1/10th, some samples will still be a bit too dilute.
Another option is to do enrichment PCR, (see my post here, but I am not sure about the primers we have or the conditions.
Any help will be greatly appreciated!
Jon
Comment