Revision as of 19:16, 20 January 2010 by >Alan6017518
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 |
999ul
|
Library DNA |
1ul
|
Total |
1000ul
|
|
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
|
|