Revision as of 18:26, 4 March 2010 by >Alan6017518
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
|
|
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]
Media:KAPA_HL042_2.pdf
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
|
|