AlanFung:Protocol/KAPA Library Quantification Kits-GA: Difference between revisions
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==KAPA Library Quantification Kits== | ==KAPA Library Quantification Kits== | ||
==For Illumina Genome Analyzer Platform== | ==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== | ==Prepare qPCR Reagents== | ||
===Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing=== | ===Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing=== | ||
Line 6: | Line 14: | ||
*6 DNA Standards | *6 DNA Standards | ||
*1:1000, 1:2000, 1:4000 & 1:8000 dilution of library DNA | *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 | |||
{| {{table}} | |||
| Library Dilution Buffer||999ul | |||
|- | |||
| Library DNA||1ul | |||
|- | |||
| Total||1000ul | |||
|- | |||
| | |||
|} | |||
1:2000 | |||
{| {{table}} | |||
| Library Dilution Buffer||100ul | |||
|- | |||
| Library DNA (1:1000)||100ul | |||
|- | |||
| Total||200ul | |||
|- | |||
| | |||
|} | |||
==qPCR Setup== | ==qPCR Setup== | ||
{| {{table}} | {| {{table}} | ||
| Setup||Volume(ul) | | Setup||Volume(ul) | ||
|- | |- | ||
| KAPA SYBR FAST qPCR Master Mix Containing Primer Premix|| | | KAPA SYBR FAST qPCR Master Mix Containing Primer Premix||39.6 | ||
|- | |- | ||
| PCR-grade water|| | | PCR-grade water||13.2 | ||
|- | |- | ||
| Diluted library DNA or DNA Standard (1-6)|| | | Diluted library DNA or DNA Standard (1-6)||13.2 | ||
|- | |- | ||
| | | | ||
|} | |} | ||
==qPCR Cycling== | ==qPCR Cycling== | ||
Step1 95C 5M | Step1 95C 5M | ||
Line 48: | Line 77: | ||
|- | |- | ||
| | | | ||
|} | |||
==Template== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- | |||
| align="center" width="65" height="12" align="center" valign="bottom" | | |||
| align="center" width="65" align="center" valign="bottom" | 1 | |||
| align="center" width="65" align="center" valign="bottom" | 2 | |||
| align="center" width="132" align="center" valign="bottom" | 3 | |||
| align="center" width="88" valign="bottom" | 4 | |||
| align="center" width="88" valign="bottom" | 5 | |||
| align="center" width="121" valign="bottom" | 6 | |||
| align="center" width="116" valign="bottom" | 7 | |||
| align="center" width="130" valign="bottom" | 8 | |||
| align="center" width="123" valign="bottom" | 9 | |||
| align="center" width="130" valign="bottom" | 110 | |||
| align="center" width="99" valign="bottom" | 11 | |||
|- | |||
| height="12" align="center" valign="bottom" | A | |||
| align="center" valign="bottom" | S1 | |||
| align="center" valign="bottom" | S3 | |||
| align="center" valign="bottom" | S6 | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-26-Sep14- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-28-Sep14- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-32-Sep14- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-Chr31-Sep14- | |||
|style="background-color:#FFFF00" valign="bottom" | JA-NA-3?????-- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-Neu4546-Sep12- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-AR3132-Sep19- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-2526-Sep7- | |||
|- | |||
| height="12" align="center" valign="bottom" | B | |||
| align="center" valign="bottom" | S1 | |||
| align="center" valign="bottom" | S4 | |||
| align="center" valign="bottom" | S6 | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-26-Sep14- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-29-Sep14- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-32-Sep14- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-Chr31-Sep14- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-Chr32,35?????-- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-Neu4546-Sep12- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-AR3132-Sep19- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | C | |||
| align="center" valign="bottom" | S1 | |||
| align="center" valign="bottom" | S4 | |||
|style="background-color:#800000" valign="bottom" | BS-NA-IVTNEX-Sep23 | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-26-Sep14- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-29-Sep14- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-2930-Sep7- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-Chr31-Sep14- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-Chr32,35?????-- | |||
|style="background-color:#0000FF" valign="bottom" | JG-NA-Nn4748-Sep12- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-AR3132-Sep19- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | D | |||
| align="center" valign="bottom" | S2 | |||
| align="center" valign="bottom" | S4 | |||
|style="background-color:#800000" valign="bottom" | BS-NA-IVTNEX-Sep23 | |||
|style="background-color:#FFFF00" valign="bottom" | JG-NA-27-Sep14- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-29-Sep14- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-2930-Sep7- | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-AR3738-Sep19- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-Chr32,35?????-- | |||
|style="background-color:#0000FF" valign="bottom" | JG-NA-Nn4748-Sep12- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-AR26?????-Sep19- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | E | |||
| align="center" valign="bottom" | S2 | |||
| align="center" valign="bottom" | S5 | |||
|style="background-color:#800000" valign="bottom" | BS-NA-IVTNEX-Sep23 | |||
|style="background-color:#FFFF00" valign="bottom" | JG-NA-27-Sep14- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-30-Sep14- | |||
|style="background-color:#660066" valign="bottom" | JG-NA-2930-Sep7- | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-AR3738-Sep19- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-Chr43????-Sep12- | |||
|style="background-color:#0000FF" valign="bottom" | JG-NA-Nn4748-Sep12- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-AR26?????-Sep19- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | F | |||
| align="center" valign="bottom" | S2 | |||
| align="center" valign="bottom" | S5 | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-25-Sep14- | |||
|style="background-color:#FFFF00" valign="bottom" | JG-NA-27-Sep14- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-30-Sep14- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-Chr4142-Sep12- | |||
|style="background-color:#FF6600" valign="bottom" | JG-NA-AR3738-Sep19- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-Chr43????-Sep12- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-Ar2930-Sep19- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-AR26?????-Sep19- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | G | |||
| align="center" valign="bottom" | S3 | |||
| align="center" valign="bottom" | S5 | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-25-Sep14- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-28-Sep14- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-30-Sep14- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-Chr4142-Sep12- | |||
|style="background-color:#FFFF00" valign="bottom" | JA-NA-3?????-- | |||
|style="background-color:#008000" valign="bottom" | JG-NA-Chr43????-Sep12- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-Ar2930-Sep19- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-2526-Sep7- | |||
| align="center" valign="bottom" | | |||
|- | |||
| height="12" align="center" valign="bottom" | H | |||
| align="center" valign="bottom" | S3 | |||
| align="center" valign="bottom" | S6 | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-25-Sep14- | |||
|style="background-color:#CCFFCC" valign="bottom" | JG-NA-28-Sep14- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-32-Sep14- | |||
|style="background-color:#800000" valign="bottom" | JG-NA-Chr4142-Sep12- | |||
|style="background-color:#FFFF00" valign="bottom" | JA-NA-3?????-- | |||
|style="background-color:#3366FF" valign="bottom" | JG-NA-Neu4546-Sep12- | |||
|style="background-color:#000090" valign="bottom" | JG-NA-Ar2930-Sep19- | |||
|style="background-color:#FF0000" valign="bottom" | JG-NA-2526-Sep7- | |||
| align="center" valign="bottom" | | |||
|} | |} |
Latest revision as of 21:06, 24 September 2012
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- |