Calendar/2010-4-27

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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 (9 samples)[edit]

Tubes Prep 9 Samples STD Total
1.5ml Tubes 9 9
0.5ml Tubes 18 6 24

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]

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[edit]

Qubit Quantification[edit]

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[edit]

Media:KAPA_HL052.pdf

Gel Quantification[edit]

File:ZhangLab 2 2010-04-27 13hr 37min.jpg

  • 37.16nM= hFib11 (350bp)
  • 57.73nM= ips11b (350bp)
  • 61.86nM= CviB (350bp)
  • 26.90nM= ips17b (350bp)
  • 74.71nM= FS (300bp)
  • 32.37nM= ips17a (350bp)
  • 67.09nM= DF (300bp)
  • 51.07nM= CV1 (350bp)
  • 84.67nM= CviF (350bp)