I always quantified DNA libraries using spectrophotometer (plate reader). Since I am using a different DNA library protocol, I want to make sure that the adapters ligated to libraries are intact and so I was planning to do qPCR on my NGS libraries. This will also help me quantify libraries accurately. So, can someone suggest me an appropriate kit or working protocol that i can use to quantify my NGS DNA libraries.
Upendra,
Although it's not very easy to quantify NGS libraries, you should try NanoDrop or a similar equipment using tiny amount of DNA and shows a detailed graph. So you can see if there is contamination or noise within your library.
Dear Upendra,
We use Kapa Library quantification kit from Kapa Biosystems. It is available for Illumina, Solid or Roche. Or universal mix.
It quatifies only the ligated molecules.
Regars
Viktor
Thanks Viktor. This is exactly what i wanted to know. Do you mind sharing your working protocol? Thanks in advance
We stuck exactly to the Kapa protocol. You find the protocols on this site: http://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/library-quantification/
Kappa Biosystems is very good for NGS LC QC. One thing that will improve your results is to use 0.1% Tween 20 for your dilutions and to vortex vigorously everything BEFORE you pipet. Three replicates is good, five is better. Downstream costs are much bigger to take shortcuts.
Kapa is indeed the way to go. You can probably play a bit with volumes and dilution numbers to reduce the overall cost. An alternative would be to try NEBNext® Library Quant Kit for Illumina® although I have myself no experience with it yet.
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