Revision as of 21:06, 24 September 2012 by >Alan6017518
KAPA Library Quantification Kits[edit]
For Illumina Genome Analyzer Platform[edit]
Prepare Library Dilution Buffer[edit]
Prepare 10mM Tris HCL, pH 8.0+0.05% Tween 20[edit]
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[edit]
Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing[edit]
- 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[edit]
- 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[edit]
- 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[edit]
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[edit]
Step1 95C 5M
Step2 95C 30S
Step3 60C 45S
Step4 Plate Read
Step5 Go to Step 2, Repeat 34X
Analysis[edit]
- 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[edit]
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
110
|
11
|
A
|
S1
|
S3
|
S6
|
JG-NA-26-Sep14-
|
JG-NA-28-Sep14-
|
JG-NA-32-Sep14-
|
JG-NA-Chr31-Sep14-
|
JA-NA-3?????--
|
JG-NA-Neu4546-Sep12-
|
JG-NA-AR3132-Sep19-
|
JG-NA-2526-Sep7-
|
B
|
S1
|
S4
|
S6
|
JG-NA-26-Sep14-
|
JG-NA-29-Sep14-
|
JG-NA-32-Sep14-
|
JG-NA-Chr31-Sep14-
|
JG-NA-Chr32,35?????--
|
JG-NA-Neu4546-Sep12-
|
JG-NA-AR3132-Sep19-
|
|
C
|
S1
|
S4
|
BS-NA-IVTNEX-Sep23
|
JG-NA-26-Sep14-
|
JG-NA-29-Sep14-
|
JG-NA-2930-Sep7-
|
JG-NA-Chr31-Sep14-
|
JG-NA-Chr32,35?????--
|
JG-NA-Nn4748-Sep12-
|
JG-NA-AR3132-Sep19-
|
|
D
|
S2
|
S4
|
BS-NA-IVTNEX-Sep23
|
JG-NA-27-Sep14-
|
JG-NA-29-Sep14-
|
JG-NA-2930-Sep7-
|
JG-NA-AR3738-Sep19-
|
JG-NA-Chr32,35?????--
|
JG-NA-Nn4748-Sep12-
|
JG-NA-AR26?????-Sep19-
|
|
E
|
S2
|
S5
|
BS-NA-IVTNEX-Sep23
|
JG-NA-27-Sep14-
|
JG-NA-30-Sep14-
|
JG-NA-2930-Sep7-
|
JG-NA-AR3738-Sep19-
|
JG-NA-Chr43????-Sep12-
|
JG-NA-Nn4748-Sep12-
|
JG-NA-AR26?????-Sep19-
|
|
F
|
S2
|
S5
|
JG-NA-25-Sep14-
|
JG-NA-27-Sep14-
|
JG-NA-30-Sep14-
|
JG-NA-Chr4142-Sep12-
|
JG-NA-AR3738-Sep19-
|
JG-NA-Chr43????-Sep12-
|
JG-NA-Ar2930-Sep19-
|
JG-NA-AR26?????-Sep19-
|
|
G
|
S3
|
S5
|
JG-NA-25-Sep14-
|
JG-NA-28-Sep14-
|
JG-NA-30-Sep14-
|
JG-NA-Chr4142-Sep12-
|
JA-NA-3?????--
|
JG-NA-Chr43????-Sep12-
|
JG-NA-Ar2930-Sep19-
|
JG-NA-2526-Sep7-
|
|
H
|
S3
|
S6
|
JG-NA-25-Sep14-
|
JG-NA-28-Sep14-
|
JG-NA-32-Sep14-
|
JG-NA-Chr4142-Sep12-
|
JA-NA-3?????--
|
JG-NA-Neu4546-Sep12-
|
JG-NA-Ar2930-Sep19-
|
JG-NA-2526-Sep7-
|
|