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==') |
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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