Hello,
I have been using the Kapa Hyper Prep kit to try and prep DNA libraries for target enrichment.
I am using custom Y adapters and dual indexed primers which I know have been used successfully with the Kapa kit before.
My issue is that I am getting virtually no amplification from the PCR. By increasing the number of cycles up to 16, my library concentrations increased by only 2-3X.
The kit I'm working with is new.
I checked my shearing with the bioanalyzer and all samples were within the targeted range (400-600 bp).
I suspect the problem is with adapter ligation...after PCR, I see very high concentrations with a nanodrop (but not Qubit) that went down sharply after bead cleanup. This makes me think lots of adapter dimers and/or unused primers were present.
Has anyone here experienced similar issues with the Kapa kit, or does anyone have any advice on how best to troubleshoot this problem?
I have been using the Kapa Hyper Prep kit to try and prep DNA libraries for target enrichment.
I am using custom Y adapters and dual indexed primers which I know have been used successfully with the Kapa kit before.
My issue is that I am getting virtually no amplification from the PCR. By increasing the number of cycles up to 16, my library concentrations increased by only 2-3X.
The kit I'm working with is new.
I checked my shearing with the bioanalyzer and all samples were within the targeted range (400-600 bp).
I suspect the problem is with adapter ligation...after PCR, I see very high concentrations with a nanodrop (but not Qubit) that went down sharply after bead cleanup. This makes me think lots of adapter dimers and/or unused primers were present.
Has anyone here experienced similar issues with the Kapa kit, or does anyone have any advice on how best to troubleshoot this problem?
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