AlanFung:Protocol/KAPA Library Quantification Kits-GA: Difference between revisions
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Step4 Plate Read | Step4 Plate Read | ||
Step5 Go to Step 2, Repeat 34X | Step5 Go to Step 2, Repeat 34X | ||
==Analysis== | |||
*Annotate the DNA standards as follows before analyzing the data according to the qPCR instrument guidelines | |||
{| {{table}} | |||
| 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 | |||
|- | |||
| | |||
|} |
Revision as of 19:01, 20 January 2010
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 |