Rui:LabNotes/Collaborations/2011-10-11: Difference between revisions
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>Sam Chiang m (Created page with '==Illumina library preparation of samples for Dr. Yi Zhang lab==') |
>RuiLiu m (→TBE gel check) |
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==Illumina library preparation of samples for Dr. | ==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: | |||
===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;"|'''''' | |||
| 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: | |||
===Concentration monitor=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''#7''' | |||
| align="center" style="background:#f0f0f0;"|'''#8''' | |||
| align="center" style="background:#f0f0f0;"|'''#9''' | |||
|- | |||
| pre-shearing||ng/ul|||||| | |||
|- | |||
| ||volume|||||| | |||
|- | |||
| ||total ng|||||| | |||
|- | |||
| after repair||ng/ul||3.03||0.665||6.63 | |||
|- | |||
| ||volume||30||30||30 | |||
|- | |||
| ||total ng||90.9||19.95||198.9 | |||
|- | |||
| after A tailing||ng/ul||3.91||1.76||8.89 | |||
|- | |||
| ||volume||18||18||18 | |||
|- | |||
| ||total ng||70.38||31.68||160.02 | |||
|- | |||
| after ligation||ng/ul||2.86||0.63||6.18 | |||
|- | |||
| ||volume||20||20||20 | |||
|- | |||
| ||total ng||57.2||12.6||123.6 | |||
|- | |||
| after PCR||ng/ul||10.6||10.6||8.47 | |||
|- | |||
| ||volume||15||15||15 | |||
|- | |||
| ||total ng||159||159||127.05 | |||
|- | |||
| ||fold change||2.77972028||12.61904762||1.027912621 | |||
|- | |||
| | |||
|- | |||
| ||pre-PCR ng||||||12 | |||
|- | |||
| post PCR||ng/ul||||||10 | |||
|- | |||
| ||volume||||||15 | |||
|- | |||
| ||total ng||||||150 | |||
|- | |||
| ||fold change||||||12.5 | |||
|} | |||
===TBE gel check=== | |||
[[File:10.12.11_RNA.jpg]] |
Latest revision as of 17:50, 13 October 2011
Illumina library preparation of samples for Dr. Xu Yang lab[edit]
DNA shearing[edit]
- Follow the protocol used on 7/8/11 [1]
- Morries Cancer Center, lab bay 2R (Yang or Sophie to contact)
End repair[edit]
- Follow the protocol used on 7/11/11 [2]
- Elute in 32ul and 1ul for Qubit measurement:
dA tailing[edit]
- Follow the protocol used on 7/11/11 [3]
- Elute in 20ul and 1ul for Qubit measurement:
Adaptor ligation[edit]
- 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[edit]
- 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:
Concentration monitor[edit]
' | Sample | #7 | #8 | #9 |
pre-shearing | ng/ul | |||
volume | ||||
total ng | ||||
after repair | ng/ul | 3.03 | 0.665 | 6.63 |
volume | 30 | 30 | 30 | |
total ng | 90.9 | 19.95 | 198.9 | |
after A tailing | ng/ul | 3.91 | 1.76 | 8.89 |
volume | 18 | 18 | 18 | |
total ng | 70.38 | 31.68 | 160.02 | |
after ligation | ng/ul | 2.86 | 0.63 | 6.18 |
volume | 20 | 20 | 20 | |
total ng | 57.2 | 12.6 | 123.6 | |
after PCR | ng/ul | 10.6 | 10.6 | 8.47 |
volume | 15 | 15 | 15 | |
total ng | 159 | 159 | 127.05 | |
fold change | 2.77972028 | 12.61904762 | 1.027912621 | |
pre-PCR ng | 12 | |||
post PCR | ng/ul | 10 | ||
volume | 15 | |||
total ng | 150 | |||
fold change | 12.5 |