Dear Friends
My name is Dr Barak Yaacov, and I am working in a medical center, we bout a Miseq to our lab and I am trying to deep sequence several genes together about 80 amplicons as a first pilot.
I decided to begin with Nextera XT kit, I design the amplicon to size between 400-900 bp. Run each one of them on gel separately clean on colons, polled together, majored by Qubit , and viewed on a bioanalyzer.
I started library prep using the NexteraXT kit fuelled all protocol lode the sample on a cartage and the run failed. At first I thought I had a problem with the normalization step. The scant time I stop after the PCR cleanup stage lode on bioanalyzer and got nothing I lost all amplicon in the way. Could it be that I need to use x1.8 AMPure XP (90ul) instead of x0.6 AMPure XP (30ul)?
When I look at the protocol of AMPure XP they recommend using x1.8 AMPure XP.
Moreover is it possible to skipped the normalization stage and after tagmentation ,PCR and clean up run the samples on bioanalyzer polled them together as in the TruSeq protocol? Since post normalization step you can't run your sample on bioanalyzer (single strand DNA) you loss an important QC step before starting the run. How much Phix should I add my sample after using the Nextera XT kit?
Many Thanks,
Barak
My name is Dr Barak Yaacov, and I am working in a medical center, we bout a Miseq to our lab and I am trying to deep sequence several genes together about 80 amplicons as a first pilot.
I decided to begin with Nextera XT kit, I design the amplicon to size between 400-900 bp. Run each one of them on gel separately clean on colons, polled together, majored by Qubit , and viewed on a bioanalyzer.
I started library prep using the NexteraXT kit fuelled all protocol lode the sample on a cartage and the run failed. At first I thought I had a problem with the normalization step. The scant time I stop after the PCR cleanup stage lode on bioanalyzer and got nothing I lost all amplicon in the way. Could it be that I need to use x1.8 AMPure XP (90ul) instead of x0.6 AMPure XP (30ul)?
When I look at the protocol of AMPure XP they recommend using x1.8 AMPure XP.
Moreover is it possible to skipped the normalization stage and after tagmentation ,PCR and clean up run the samples on bioanalyzer polled them together as in the TruSeq protocol? Since post normalization step you can't run your sample on bioanalyzer (single strand DNA) you loss an important QC step before starting the run. How much Phix should I add my sample after using the Nextera XT kit?
Many Thanks,
Barak
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