Hi all,
We are doing the metagenomics study by using RT-PCR products as the samples for the libraries preparation. We are using NEBNext Quick DNA Sample Prep kit for the job.
We have selected the DNA fragments from 300bp to 800bp by gel method first, then we proceeded the library preparation as referred to NEB instruction manual. We have used Roche MID adaptors for the ligation step. We have done the qPCR (KAPA) to quantify the amount of molecules with adaptors properly ligated. Unfortunately, we found the yield (the amount of molecules with ligated adaptors) was poor which were not enough for downstream LV emPCR for FLX. Compared with sample from sheared genomic DNA for library preparation, the amount of the molecules from RT-PCR products were lower than in 10 to 100 fold.
We wonder anyone have similar experience and how to get through the problem. Did you think the method to prepare RT-PCR product can affect the ligation efficiency?
Thanks for your input
We are doing the metagenomics study by using RT-PCR products as the samples for the libraries preparation. We are using NEBNext Quick DNA Sample Prep kit for the job.
We have selected the DNA fragments from 300bp to 800bp by gel method first, then we proceeded the library preparation as referred to NEB instruction manual. We have used Roche MID adaptors for the ligation step. We have done the qPCR (KAPA) to quantify the amount of molecules with adaptors properly ligated. Unfortunately, we found the yield (the amount of molecules with ligated adaptors) was poor which were not enough for downstream LV emPCR for FLX. Compared with sample from sheared genomic DNA for library preparation, the amount of the molecules from RT-PCR products were lower than in 10 to 100 fold.
We wonder anyone have similar experience and how to get through the problem. Did you think the method to prepare RT-PCR product can affect the ligation efficiency?
Thanks for your input