AlanFung:Protocol/KAPA Library Quantification Kits-GA: Difference between revisions
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Revision as of 18:40, 20 September 2012
KAPA Library Quantification Kits
For Illumina Genome Analyzer Platform
Prepare Library Dilution Buffer
Prepare 10mM Tris HCL, pH 8.0+0.05% Tween 20
Need 1200ul for 1 sample
- Make about 20ml
- 10ml*0.05%=5ul Tween20
- Mix 10ml 10mM Tris HCL with 5ul Tween 20
- Mix 6.4ml 31.25mM Tric-HCL with 13.6ml ddH2O and add in 10ul 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 |
1:2000
Library Dilution Buffer | 100ul |
Library DNA (1:1000) | 100ul |
Total | 200ul |
qPCR Setup
Setup | Volume(ul) |
KAPA SYBR FAST qPCR Master Mix Containing Primer Premix | 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 |
Template
1 | 2 | 3 | 4 | |
A | S1 | S3 | S6 | BS-Mixed-Sep17 |
B | S1 | S4 | S6 | BS-Mixed-Sep17 |
C | S1 | S4 | JG-Ind32_33-Aug28 | BS-Mixed-Sep17 |
D | S2 | S4 | JG-Ind32_33-Aug28 | |
E | S2 | S5 | JG-Ind32_33-Aug28 | |
F | S2 | S5 | JG-Ind47_48-Aug28 | |
G | S3 | S5 | JG-Ind47_48-Aug28 | |
H | S3 | S6 | JG-Ind47_48-Aug28 |