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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 77: Line 77:
|-
|-
|  
|  
|}
==Template==
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|-  align="center" valign="bottom"
| align="center" width="132" height="12" | &nbsp;
| align="center" width="65" | 1
| align="center" width="65" | 2
| align="center" width="132" | 3
| align="center" width="129" | 4
|-  align="center" valign="bottom"
| height="12" | A
| S1
| S3
| S6
|style="background-color:#FFFF00" | BS-Mixed-Sep17
|-  align="center" valign="bottom"
| height="12" | B
| S1
| S4
| S6
|style="background-color:#FFFF00" | BS-Mixed-Sep17
|-  align="center" valign="bottom"
| height="15" | C
| S1
| S4
|style="background-color:#FF6600;font-size:12pt" | JG-Ind32_33-Aug28
|style="background-color:#FFFF00" | BS-Mixed-Sep17
|-  align="center" valign="bottom"
| height="15" | D
| S2
| S4
|style="background-color:#FF6600;font-size:12pt" | JG-Ind32_33-Aug28
| align="center" | &nbsp;
|-  align="center" valign="bottom"
| height="15" | E
| S2
| S5
|style="background-color:#FF6600;font-size:12pt" | JG-Ind32_33-Aug28
| align="center" | &nbsp;
|-  align="center" valign="bottom"
| height="15" | F
| S2
| S5
|style="background-color:#FF6600;font-size:12pt" | JG-Ind47_48-Aug28
| align="center" | &nbsp;
|-  align="center" valign="bottom"
| height="15" | G
| S3
| S5
|style="background-color:#FF6600;font-size:12pt" | JG-Ind47_48-Aug28
| align="center" | &nbsp;
|-  align="center" valign="bottom"
| height="15" | H
| S3
| S6
|style="background-color:#FF6600;font-size:12pt" | JG-Ind47_48-Aug28
| align="center" | &nbsp;
|}
|}

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