Calendar/2010-4-27: Difference between revisions
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===QPCR Quantification=== | ===QPCR Quantification=== | ||
[[Media:KAPA_HL052.pdf]] | |||
===Gel Quantification=== | ===Gel Quantification=== |
Revision as of 16:17, 29 April 2010
Quantification of Solexa Sequencing Libraries using KAPA Quantification Kits
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 31.25nM Tric-HCL with 13.6ml ddH2O and add in 10ul tween-20
Prepare Tubes (9 samples)
Tubes Prep | 9 Samples | STD | Total |
1.5ml Tubes | 9 | 9 | |
0.5ml Tubes | 18 | 6 | 24 |
Make 1:1000 Dilution
- 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
Setup | ||||||
1 | 2 | 3 | 4 | 5 | 6 | |
A | S1 | S3 | S6 | AL-Exome-CViB-Apr23 | AL-Exome-FS-Apr23 | AL-Exome-CV1-Apr23 |
B | S1 | S4 | S6 | AL-Exome-CViB-Apr23 | AL-Exome-ips17a-Apr23 | AL-Exome-CV1-Apr23 |
C | S1 | S4 | AL-Exome-hFib11-apr23 | AL-Exome-CViB-Apr23 | AL-Exome-ips17a-Apr23 | AL-Exome-CViF-Apr25 |
D | S2 | S4 | AL-Exome-hFib11-apr23 | AL-Exome-ips17b-Apr23 | AL-Exome-ips17a-Apr23 | AL-Exome-CViF-Apr25 |
E | S2 | S5 | AL-Exome-hFib11-apr23 | AL-Exome-ips17b-Apr23 | AL-Exome-DF-Apr23 | AL-Exome-CViF-Apr25 |
F | S2 | S5 | AL-Exome-ips11b-apr23 | AL-Exome-ips17b-Apr23 | AL-Exome-DF-Apr23 | |
G | S3 | S5 | AL-Exome-ips11b-apr23 | AL-Exome-FS-Apr23 | AL-Exome-DF-Apr23 | |
H | S3 | S6 | AL-Exome-ips11b-apr23 | AL-Exome-FS-Apr23 | AL-Exome-CV1-Apr23 | |
- Load 20ul to each well
Results
Qubit Quantification
Invitrogen | ||||||
Qubit(TM) Fluorometer | ||||||
04/28/10 10:52 AM | ||||||
Quant-iT dsDNA | BR | |||||
Sample | Concentration in the Qubit | uL used | Dilution | Sample Concentration | ||
AL-Exome-hFib11-apr23 | 0.06 | ug/mL | 1 | 200 | 11.3 | ng/ul |
AL-Exome-ips11b-apr23 | 0.08 | ug/mL | 1 | 200 | 16.6 | ng/ul |
AL-Exome-CViB-Apr23 | 0.11 | ug/mL | 1 | 200 | 22.8 | ng/ul |
AL-Exome-ips17b-Apr23 | 0.04 | ug/mL | 1 | 200 | 7.05 | ng/ul |
AL-Exome-FS-Apr23 | 0.12 | ug/mL | 1 | 200 | 23 | ng/ul |
AL-Exome-ips17a-Apr23 | 0.05 | ug/mL | 1 | 200 | 11 | ng/ul |
AL-Exome-DF-Apr23 | 0.08 | ug/mL | 1 | 200 | 15.7 | ng/ul |
AL-Exome-CV1-Apr23 | 0.08 | ug/mL | 1 | 200 | 15.1 | ng/ul |
AL-Exome-CViF-Apr25 | 0.18 | ug/mL | 1 | 200 | 35.5 | ng/ul |