Rui:LabNotes/Collaborations/2011-10-11: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang |
||
Line 1: | Line 1: | ||
==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 |