AlanFung:Protocol/KAPA Library Quantification Kits-GA

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Revision as of 18:29, 28 March 2010 by >Alan6017518 (→‎qPCR Setup)
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KAPA Library Quantification Kits

For Illumina Genome Analyzer Platform

Prepare Library Dilution Buffer

  • 10mM Tris HCL, pH 8.0+0.0.5% Tween 20

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

Prepare qPCR Master Mix

  • Add 1ml of Illumina GA Primer Premix(10X) to 5ml bottle of KAPA SYBR FAST qPCR Master Mix (2X) and mix well

Perform Serial Dilution of dsDNA Libraries

  • Dilute purified library DNA in 10mM Tris HCL, pH 8.0+0.0.5% Tween 20 to 1:1000, 1:2000, 1:4000, 1:8000 dilution.
1:1000
Library Dilution Buffer 998ul
Library DNA 2ul
Total 1000ul
1:2000
Library Dilution Buffer 100ul
Library DNA (1:1000) 100ul
Total 200ul

qPCR Setup

Setup Volume(ul)
39.6
PCR-grade water 13.2
Diluted library DNA or DNA Standard (1-6) 13.2

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