Rui:LabNotes/Collaborations/2011-10-12: Difference between revisions

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>Sam Chiang
m (Created page with '==Illumina library preparation of samples for Dr. Yi Zhang lab==')
 
>RuiLiu
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==Illumina library preparation of samples for Dr. Yi Zhang lab==
==Illumina library preparation of samples for Dr. Yi Zhang lab==
===DNA shearing===
* Follow the protocol used on 7/8/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-8]
* Morries Cancer Center, lab bay 2R (Yang or Sophie to contact)
===End repair===
* Follow the protocol used on 7/11/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#End_repair]
* Elute in 32ul and 1ul for Qubit measurement:
===dA tailing===
* Follow the protocol used on 7/11/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#A_tailing]
* Elute in 20ul and 1ul for Qubit measurement:
===Adaptor ligation===
* Follow the protocol used on 7/11/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#Adapter_ligation]
* 20uM fresh made adaptor, ~1:20 ratio
* Calculation for adaptor input:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''samples'''
| align="center" style="background:#f0f0f0;"|'''ng/ul'''
| align="center" style="background:#f0f0f0;"|'''volume'''
| align="center" style="background:#f0f0f0;"|'''total ng'''
| align="center" style="background:#f0f0f0;"|'''pmole'''
| align="center" style="background:#f0f0f0;"|'''adaptor'''
| align="center" style="background:#f0f0f0;"|'''ul'''
| align="center" style="background:#f0f0f0;"|'''adjusted ul'''
|-
| ||||||||||||||
|-
| 1||3.28||18||59.04||0.4542||9.0831||0.4542||0.8
|-
| ||||||||||||||
|-
| 2||3.55||18||63.9||0.4915||9.8308||0.4915||0.8
|-
| ||||||||||||||
|-
| 3||4.75||18||85.5||0.6577||13.1538||0.6577||1
|-
| ||||||||||||||
|-
| 4||5.16||18||92.88||0.7145||14.2892||0.7145||1
|-
| ||||||||||||||
|-
| 5||2.44||18||43.92||0.3378||6.7569||0.3378||0.5
|-
| ||||||||||||||
|-
| 6||0.39||18||6.93||0.0533||1.0662||0.0533||0.5
|}
===PCR amplification===
* After ligation and beads purification, 22ul EB is used to elute DNA, take 20ul into tubes, take 5ul of s1-5 for PCR, use all (20ul) of s6
* 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 10 cycles
* beads purification: 0.7X, elute in 15ul, take 12ul

Revision as of 17:47, 13 October 2011

Illumina library preparation of samples for Dr. Yi Zhang lab

DNA shearing

  • Follow the protocol used on 7/8/11 [1]
  • Morries Cancer Center, lab bay 2R (Yang or Sophie to contact)

End repair

  • Follow the protocol used on 7/11/11 [2]
  • Elute in 32ul and 1ul for Qubit measurement:

dA tailing

  • Follow the protocol used on 7/11/11 [3]
  • Elute in 20ul and 1ul for Qubit measurement:

Adaptor ligation

  • Follow the protocol used on 7/11/11 [4]
  • 20uM fresh made adaptor, ~1:20 ratio
  • Calculation for adaptor input:
samples ng/ul volume total ng pmole adaptor ul adjusted ul
1 3.28 18 59.04 0.4542 9.0831 0.4542 0.8
2 3.55 18 63.9 0.4915 9.8308 0.4915 0.8
3 4.75 18 85.5 0.6577 13.1538 0.6577 1
4 5.16 18 92.88 0.7145 14.2892 0.7145 1
5 2.44 18 43.92 0.3378 6.7569 0.3378 0.5
6 0.39 18 6.93 0.0533 1.0662 0.0533 0.5

PCR amplification

  • After ligation and beads purification, 22ul EB is used to elute DNA, take 20ul into tubes, take 5ul of s1-5 for PCR, use all (20ul) of s6
  • 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 10 cycles
  • beads purification: 0.7X, elute in 15ul, take 12ul