GeCKOv2 amplicon library (PCR1 and PCR2 products) quantification

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?

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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?

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Your thoughts and suggestions will be much appreciated.


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Fuchsia Kelpieover 3 years ago

I'm not sure which primer sets you are using, but I have seen in a different post on this google forum that the qPCR quantification produces very low yields relative to the actual yield. You should not need to clean up PCR1 product before performing PCR2 if you are using 1-2uL of PCR1 product for PCR2 input.

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