problems with emPCR for GS Junior

[Vartul]

Hi All,

I have an unusual problem with the consistency of emPCR kits that were received two months ago (emPCR oil and breaking kit (Lot: 93896820), emPCR Lib-L kit (lot: 93896420) and Bead recovery kit (lot: 93893620). Of the eight kits, three worked well but five emPCRs have failed.

We calculate the copy number using the library concentration from Qubit and average size from the bioanalyzer, similar to what Roche suggested for quantification of pair-end libraries. This method has worked well in past as it has worked for three emPCRs from this batch. We also run a normal PCR reaction to test the library amplificability which suggest that the libraries are fine.

I feel the problem is with the emPCR kits because:
1. We had a gloppy thing floating over the beads during the bead recovery three times it failed. It appears to be the problem with emulsion oil.
2. I repeated the emPCR for the library that has worked before (using same amount) but it failed the second time.
3. I tripled the amount of library in the emPCR (as suggested in the emPCR manual) but still not enough beads were recovered.

I was wondering if someone else experienced similar issue. Any suggestions will be highly appreciated.

Thanks and regards,