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]

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