Calendar/2010-3-2
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Quantification of Solexa Sequencing Libraries using KAPA Quantification Kits[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 31.25nM Tric-HCL with 13.6ml ddH2O and add in 10ul tween-20
Prepare Tubes[edit]
Tubes Prep | 2 Samples | STD | Total |
1.5ml Tubes | 2 | 2 | |
0.5ml Tubes | 2 | 6 | 8 |
Make 1:1000 Dilution[edit]
- Label tubes (1 nonstick 1.5ml tube + 2 nonstick 0.5ml tubes)x5 + 6 0.5ml tubes for standard
- Dilute purified library DNA in 10mM Tris HCL, pH 8.0+0.0.5% Tween 20 to 1:1000 dilution
1:500
Library Dilution Buffer | 998ul |
Library DNA | 2ul |
Total | 1000ul |
1:1000
Library Dilution Buffer | 100ul |
Library DNA | 100ul |
Total | 200ul |
qPCR Setup[edit]
X3.3 | |
KAPA SYBR FAST qPCR Master Mix Containing Primer Premix | 39.6 |
PCR-grade water | 13.2 |
Library/Standard | 13.2 |
- Load 20ul to each well
Setup | |||
1 | 2 | 3 | |
A | S1 | S3 | S6 |
B | S1 | S4 | S6 |
C | S1 | S4 | AL-DigiQ-Gol+GSG+SoR |
D | S2 | S4 | AL-DigiQ-Gol+GSG+SoR |
E | S2 | S5 | AL-DigiQ-Gol+GSG+SoR |
F | S2 | S5 | AL-Exome-CViB-Feb21 |
G | S3 | S5 | AL-Exome-CViB-Feb21 |
H | S3 | S6 | AL-Exome-CViB-Feb21 |
Results[edit]
Gel Quantification[edit]
File:ZhangLab 2 2010-02-24 11hr 16min-CViB.jpg
Size Adjustment | Gel | Gel | Gel/QPCR | Load | |||
Sample | nM | BP | nM | ng/ul | nM | Ratio | pM |
AL-Exome-CViB-Feb21 | 2.71 | 300 | 4.08 | N/A | 197 | 48.25 | 2 |