Calendar/2010-4-27: Difference between revisions

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>Alan6017518
(Created page with '==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…')
 
>Alan6017518
 
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==Results==
==Results==
==Qubit Quantification==
{| {{table}}
| 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===
===QPCR Quantification===
 
[[Media:KAPA_HL052.pdf]]


===Gel Quantification===
===Gel Quantification===
[[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)

Latest revision as of 16:19, 29 April 2010

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)