We have been having trouble getting good libraries for the last few weeks. After doing the library prep, the libraries look just fine on the Bioanalyzer and the concentration is also right where it should be. Then, we do the emPCR and we get no beads at all.
This is true of every library we've made in the last 3-4 weeks. There are 4 that we made first and they had this problem. Another person remade two of them and those to did the same thing. In the mean time, before we figured out it was a library prep problem, he made 24 more libraries. Testing of a few of them showed the same results. We also had a demo of a robot to automate the library prep, and the libraries made by the robot also looked great but didn't work in emPCR. In the meantime, I ran some emulsions with one of these new libraries and an old library that had already been sequenced. The old one worked worked just fine but the new one failed. Finally, these libraries were prepared from library prep kits from two different lots.
We've ordered new library prep kits but they seem to be on backorder.
I don't know for sure what's wrong with these libraries, but my best guess is that the kinase didn't work. If that were the case, the adapters would be ligated on one strand but not the other. This would make them quantify fine and look fine on the Bioanalyzer, but they would fail in emPCR.
Has anyone else had trouble of this sort? Any other ideas what could be the problem and how to solve it?
This is true of every library we've made in the last 3-4 weeks. There are 4 that we made first and they had this problem. Another person remade two of them and those to did the same thing. In the mean time, before we figured out it was a library prep problem, he made 24 more libraries. Testing of a few of them showed the same results. We also had a demo of a robot to automate the library prep, and the libraries made by the robot also looked great but didn't work in emPCR. In the meantime, I ran some emulsions with one of these new libraries and an old library that had already been sequenced. The old one worked worked just fine but the new one failed. Finally, these libraries were prepared from library prep kits from two different lots.
We've ordered new library prep kits but they seem to be on backorder.
I don't know for sure what's wrong with these libraries, but my best guess is that the kinase didn't work. If that were the case, the adapters would be ligated on one strand but not the other. This would make them quantify fine and look fine on the Bioanalyzer, but they would fail in emPCR.
Has anyone else had trouble of this sort? Any other ideas what could be the problem and how to solve it?
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