AlanFung:Protocol/KAPA Library Quantification Kits-GA

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Revision as of 19:06, 20 January 2010 by >Alan6017518
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KAPA Library Quantification Kits

For Illumina Genome Analyzer Platform

Prepare qPCR Reagents

Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing

  • 2X KAPA SYBR FAST qPCR Master Mix
  • 6 DNA Standards
  • 1:1000, 1:2000, 1:4000 & 1:8000 dilution of library DNA

Step1

  • Perform an an 1:1000 dilution of the purified library in Library Dilution Buffer
  • Optional 1:2000, 1:4000, 1:8000

Step2

qPCR Setup

Setup Volume(ul)
KAPA SYBR FAST qPCR Master Mix Containing Primer Premix 12
PCR-grade water 4
Diluted library DNA or DNA Standard (1-6) 4

qPCR Cycling

Step1 95C 5M
Step2 95C 30S
Step3 60C 45S
Step4 Plate Read
Step5 Go to Step 2, Repeat 34X

Analysis

  • Annotate the DNA standards as follows before analyzing the data according to the qPCR instrument guidelines
Sample Name dsDNA Concentration (pM)
Std 1 20
Std 2 2
Std 3 0.2
Std 4 0.02
Std 5 0.002
Std 6 0.0002