AlanFung:Protocol/KAPA Library Quantification Kits-GA: Difference between revisions

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==For Illumina Genome Analyzer Platform==
==For Illumina Genome Analyzer Platform==
==Prepare Library Dilution Buffer==
==Prepare Library Dilution Buffer==
*10nM Tris HCL, pH 8.0+0.0.5% Tween 20
==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==
Line 13: Line 18:


==Perform Serial Dilution of dsDNA Libraries==
==Perform Serial Dilution of dsDNA Libraries==
*Dilute library DNA in 10
*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.
*Perform an 1:1000, 1:2000, 1:4000, 1:8000 dilution of the purified library in Library Dilution Buffer
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==
Line 20: Line 44:
| Setup||Volume(ul)
| Setup||Volume(ul)
|-
|-
| KAPA SYBR FAST qPCR Master Mix Containing Primer Premix||12
| KAPA SYBR FAST qPCR Master Mix Containing Primer Premix||39.6
|-
|-
| PCR-grade water||4
| PCR-grade water||13.2
|-
|-
| Diluted library DNA or DNA Standard (1-6)||4
| Diluted library DNA or DNA Standard (1-6)||13.2
|-
|-
|  
|  
|}
|}
==qPCR Cycling==
==qPCR Cycling==
  Step1 95C 5M
  Step1 95C 5M
Line 52: 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-