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Old 07-23-2014, 03:44 AM   #3
Location: Netherlands

Join Date: Jul 2013
Posts: 13


I have been using the TruSeq DNA/TruSeq Nano kit on degraded FFPE material for quite some time now. For the FFPE material we use 150ng qubit measured DNA as input and we know from experience that this gives us good results.
As we are using samples below the optimal standard, we have found that diluting the adapters 1:9 helps (use 5 ul of that dilution in the procedure). For FFPE material we add all of the cleaned-up ligation product into the pcr reaction (for good quality DNA we use half) and we run the pcr with 10 cycles (as for good quality DNA we only do 8 cycles).
After sampleprep we always measure the sample with the BioAnalyzer, but with low quality samples there might be problems with adapter-dimers in the final prep result (this is the reason we also dilute the adapter, but sometimes it doesn't help). If this is the case we do an AMPure XP beads cleanup and measure again before running on the MiSeq/HiSeq.

** update 20140724**
I did perform the prep with end-repair, but as I did not change anything in that part of the protocol I did not mention it.

Last edited by WhiteSeal; 07-24-2014 at 04:34 AM.
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