Quantification of DNA Sequencing Libraries for Next Generation Sequencing in Illumina Sequencing Platform

August 12, 2016

University of Birmingham
Biosciences
Research Assistant

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Quality of Results

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Company:

KAPA Biosystems

Product Name:

Illumina Library Quantification Kit Universal qPCR mix

Catalog Number:

KK4824

Universal qPCR mix from Kapa systems was used to quantify the DNA sequencing libraries prepared for Illumina MiSeq platform. Kit performed very well with generation linear standard curve (R^2 of 0.99) and consistent quantification values among the libraries. Based in this quantification, I used 20 pM libraries for sequencing and it resulted in optimal cluster generation in the MiSeq.

Experimental Design and Results Summary

Application

Quantification of Sequencing libraries for Illumina sequencing platforms

Starting Material

Salmonella enterica DNA sequencing libraries prepared using NEBNext Ultra library kit

Protocol Overview

Prepared and loaded DNA standards provided in the kit and loaded then into 96-well microplate in 20 microliter reaction volume. Then sequencing libraries were diluted in 10 fold and diluted samples were loaded into 96-well microplate (each dilution with triplicate). Cycling conditions, data collection and quantification analysis were performed according to the kit's instructions.

Tips

None

Results Summary

Run went well and generated very good standard curve for DNA standards (R^2 of 0.99). Most of the libraries prepared were had 50 nM concentration.

Additional Notes

None

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Summary

The Good

Protocol for performing qPCR quantification is very simple and easy to follow. No hassles of complexity.

The Bad

None

The Bottom Line

qPCR based quantification of sequencing libraries is must step prior to loading your samples onto sequencing platform. To do that, KAPA biosystems qPCR master mix is ideal.

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