Rui:LabNotes/Collaborations/2011-10-11: Difference between revisions

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>Sam Chiang
>Sam Chiang
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==Illumina library preparation of samples for Dr. Xu Yang lab==
==Illumina library preparation of samples for Dr. Xu Yang 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:
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Concentration'''
| align="center" style="background:#f0f0f0;"|'''ng/ul'''
|-
| 7||||ug/mL
|-
| 8||||ug/mL
|-
| 9||||ug/mL
|-
|
|}
===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:
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Concentration'''
| align="center" style="background:#f0f0f0;"|'''ng/ul'''
|-
| 7||||ug/mL
|-
| 8||||ug/mL
|-
| 9||||ug/mL
|-
|
|}
===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;"|''''''
| 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;"|'''add'''
|-
| #7||3.91||18||70.38||0.5414||10.83||0.54||0.5
|-
| #8||1.76||18||31.68||0.2437||4.87||0.24||0.5
|-
| #9||8.89||18||160.02||1.2309||24.62||1.23||1.2
|-
|
|}
===PCR amplification===
* After ligation and beads purification, 25ul EB is used to elute DNA, 23ul to PCR
* 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 8 cycles
* Pre- and post-PCR measurement:
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''ng/ul'''
| align="center" style="background:#f0f0f0;"|'''volume'''
| align="center" style="background:#f0f0f0;"|'''total'''
|-
| pre-7||2.86||20||57.2
|-
| post-7||10.6||15||159
|-
| pre-8||0.63||20||12.6
|-
| post-8||10.6||15||159
|-
| pre-9||6.18||20||123.6
|-
| post-9||8.47||15||127.05
|-
|
|}

Revision as of 02:19, 13 October 2011

Illumina library preparation of samples for Dr. Xu Yang 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:
Samples Concentration ng/ul
7 ug/mL
8 ug/mL
9 ug/mL

dA tailing

  • Follow the protocol used on 7/11/11 [3]
  • Elute in 20ul and 1ul for Qubit measurement:
Samples Concentration ng/ul
7 ug/mL
8 ug/mL
9 ug/mL

Adaptor ligation

  • Follow the protocol used on 7/11/11 [4]
  • 20uM fresh made adaptor, ~1:20 ratio
  • Calculation for adaptor input:
' ng/ul volume total ng pmole adaptor ul add
#7 3.91 18 70.38 0.5414 10.83 0.54 0.5
#8 1.76 18 31.68 0.2437 4.87 0.24 0.5
#9 8.89 18 160.02 1.2309 24.62 1.23 1.2

PCR amplification

  • After ligation and beads purification, 25ul EB is used to elute DNA, 23ul to PCR
  • 50ul PCR reaction: 25ul Kapa HF, 1ul N2.F, 1ul N2.R7-15, 0.5ul 20X SYBR green; 8 cycles
  • Pre- and post-PCR measurement:
' ng/ul volume total
pre-7 2.86 20 57.2
post-7 10.6 15 159
pre-8 0.63 20 12.6
post-8 10.6 15 159
pre-9 6.18 20 123.6
post-9 8.47 15 127.05