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

QPCR Quantification

Media:KAPA_HL052.pdf

Gel Quantification