I prepared amplicon libraries from the plasmid DNA (GeCKOv2 library plasmid) and genomic DNA. I used PCR1 product in the PCR2 reaction directly without any clean-up of PCR1 products.
It showed the correct band size (~367bp) of the PCR2 product when ran on the agarose gel, but bigger (~550bp) on the Bioanalyzer. In addition, there was almost no library present when quantified by the core facility using qPCR. The core facility used Illumina primers specific to the “P5” and “P7” sequence which should be present in all Illumina sequence libraries. I am not sure whether Illumina primers specific to the “P5” and “P7” should also work for these amplicon libraries practically.
Now my questions are.......
Has anyone quantified these amplicon libraries using qPCR?
If I clean-up PCR1 product and then use it for the PCR2 reaction, will it help? If yes, should I do regular PCR product clean-up or I should run on the gel to extract the correct band?
Your thoughts and suggestions will be much appreciated.