Starting with 10ug of high quality total RNA (via BA) we still seem to squeak by having enough to pass the qtPCR step at the sequencing lab. We have tried to titrate the adapter/fragment ratio to 10:1 but that hasn't helped. Typically, 30-60ng pre-ligation step, 2x is much at QC before end repair, normal numbers? Follow the Illumina mRNA seq protocol to the tee but add a second gel extraction step after the enrichment PCR of 15 cycles to help with de novo assembly.
What kinds of concentrations are people getting?
5-20ng library concentration via 1000bp BA run at the end but still not passing qtPCR, why not? what is coming thru the gel extraction?
What can we do to improve library concentration? We typically only have 1 shot at the libraries and some don't make it all the way thru to the end. Any step to go back and try again?
We eventually want to normalize the libraries but not with these concentrations.
Thanks for the help.
What kinds of concentrations are people getting?
5-20ng library concentration via 1000bp BA run at the end but still not passing qtPCR, why not? what is coming thru the gel extraction?
What can we do to improve library concentration? We typically only have 1 shot at the libraries and some don't make it all the way thru to the end. Any step to go back and try again?
We eventually want to normalize the libraries but not with these concentrations.
Thanks for the help.