Hello All
We are doing the Sureselect protocol (XT all exon V4) with 3ug of DNA, after size selection we have around 1ug left for the end repair, A addition, adaptors and cleaning etc. Half of that then goes into the 6 cycles of PCR and we often have around 20uls x 30ng of DNA which is barely enough to go into the hyb.
Is anyone seeing better concentrations at this stage or is that as good as it gets?
Post hyb, after amplification we also have smallish yield (normally just enough for 1 run) is it worth doing more than the recommended 8 cycles of PCR at this stage so that we have library to come back to if we need to repeat or top up the sequence data? What concentration do you normally see after this 8 cycle PCR?
Thanks
JPC
We are doing the Sureselect protocol (XT all exon V4) with 3ug of DNA, after size selection we have around 1ug left for the end repair, A addition, adaptors and cleaning etc. Half of that then goes into the 6 cycles of PCR and we often have around 20uls x 30ng of DNA which is barely enough to go into the hyb.
Is anyone seeing better concentrations at this stage or is that as good as it gets?
Post hyb, after amplification we also have smallish yield (normally just enough for 1 run) is it worth doing more than the recommended 8 cycles of PCR at this stage so that we have library to come back to if we need to repeat or top up the sequence data? What concentration do you normally see after this 8 cycle PCR?
Thanks
JPC
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