Rui:LabNotes/Collaborations/2011-7-11: Difference between revisions
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>Dinh |
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'''Purification with AMPure beads''' | '''Purification with AMPure beads''' | ||
# | # 150ul beads to 100ul reaction (kapa lib prep kit suggested), mix 10 times, R.T for 8min | ||
# Place on magnetic stand for 5min | # Place on magnetic stand for 5min | ||
# Remove sup., wash with 180ul fresh made 80% EtOH twice | # Remove sup., wash with 180ul fresh made 80% EtOH twice | ||
# Air-dry for 15min, re-suspend with 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | # Air-dry for 15min, re-suspend with 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | ||
# Transfer 30ul DNA from beads | # Transfer 30ul DNA from beads | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''After end''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ng''' | |||
|- | |||
| GFP+||0.35||10.5 | |||
|- | |||
| GFP-||0.24||7.2 | |||
|- | |||
| E9.5||0.39||11.7 | |||
|- | |||
| E11.5||1.29||38.7 | |||
|- | |||
| E13.5m||0.54||16.2 | |||
|- | |||
| E13.5f||2.27||68.1 | |||
|- | |||
| | |||
|} | |||
===A tailing=== | ===A tailing=== | ||
'''Reaction''' | |||
End-repaired DNA 30ul | |||
Master mix buffer 5ul | |||
Enzyme mix 3ul | |||
H2O 12ul | |||
'''Total 50ul''' | |||
37C for 30min | |||
'''Purification with AMPure beads''' | |||
# 90ul beads to 50ul reaction, mix 10 times, R.T for 8min | |||
# Place on magnetic stand for 5min | |||
# Remove sup., wash with 180ul fresh made 80% EtOH twice | |||
# Air-dry for 15min, re-suspend with 20ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | |||
# Transfer 18ul DNA from beads, 1ul left in tube for Qubit measurement | |||
===Adapter ligation=== | ===Adapter ligation=== | ||
* 30uM Y-adapter made by Alice few weeks ago | |||
* 0.08ul for sample 1,3,4,5,7 and 0.4ul for 2,6,8 | |||
* 50ul total with 10ul buffer and 5ul Quick T4 ligase; RT for 30min | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ng''' | |||
| align="center" style="background:#f0f0f0;"|'''ug''' | |||
| align="center" style="background:#f0f0f0;"|'''pmole''' | |||
| align="center" style="background:#f0f0f0;"|'''adaptor''' | |||
| align="center" style="background:#f0f0f0;"|'''ul (30uM)''' | |||
|- | |||
| PC2||0.91||16.4||0.016||0.12||2.42||0.08 | |||
|- | |||
| GFP+||0.29||5.27||0.005||0.04||0.76||0.03 | |||
|- | |||
| GFP-||0.23||4.14||0.004||0.03||0.6||0.02 | |||
|- | |||
| E9.5||0.4||7.22||0.007||0.05||1.06||0.04 | |||
|- | |||
| E11.5||1.61||28.98||0.029||0.22||4.4||0.15 | |||
|- | |||
| E13.5m||0.48||8.59||0.009||0.07||1.36||0.05 | |||
|- | |||
| E13.5f||2.72||48.96||0.049||0.37||7.42||0.25 | |||
|- | |||
| | |||
|} | |||
'''Purification with AMPure beads''' | |||
# 50ul beads to 50ul reaction, mix 10 times, R.T for 8min | |||
# Place on magnetic stand for 5min | |||
# Remove sup., wash with 180ul fresh made 80% EtOH twice | |||
# Air-dry for 15min, re-suspend with 22ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | |||
# Transfer 20ul DNA from beads, 1ul left in tube for Qubit measurement | |||
===PCR amplification=== | ===PCR amplification=== | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ng''' | |||
|- | |||
| PC1||0.15||2.7 | |||
|- | |||
| PC2||0.87||15.61 | |||
|- | |||
| GFP+||0.25||4.52 | |||
|- | |||
| GFP-||0.24||4.3 | |||
|- | |||
| E9.5||0.31||5.51 | |||
|- | |||
| E11.5||1.22||21.96 | |||
|- | |||
| E13.5m||0.51||9.22 | |||
|- | |||
| E13.5f||2.63||47.34 | |||
|- | |||
| | |||
|} | |||
====Pre-run==== | |||
* Pre-run with PC1 (lowest amount of template) and PC2 (relatively high amount of template) | |||
* 50ul total: 25ul Phusion master mix, 1ul 10uM PCR primer each | |||
* 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (15 cycles); 72C 5min | |||
[[File:7.11.11_pre-run.jpg]] | |||
[[File:7.11.11_pre-run gel check.jpg]] | |||
Something wrong with the loading buffer, use a new one! | |||
====Sample run==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ng''' | |||
|- | |||
| PC1||0.15||18||2.7 | |||
|- | |||
| PC2||0.87||18||15.61 | |||
|- | |||
| GFP+||0.25||10||2.5 | |||
|- | |||
| GFP-||0.24||10||2.4 | |||
|- | |||
| E9.5||0.31||10||3.1 | |||
|- | |||
| E11.5||1.22||2||2.4 | |||
|- | |||
| E13.5m||0.51||5||2.5 | |||
|- | |||
| E13.5f||2.63||1||2.6 | |||
|- | |||
| | |||
|} | |||
* 50ul total: 25ul Phusion master mix; 1ul 10uM Pf; 1ul 10uM Pr.ID1 to 6 | |||
* 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (12 cycles); 72C 5min | |||
QPCR curve | |||
[[File:7.11.11_qpcr.jpg]] | |||
===Size selection=== | ===Size selection=== | ||
Continue with lab note on 7.12.11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-12] |
Latest revision as of 22:21, 2 September 2011
Prepare RNAseq library for sequencing[edit]
- Continue with lab note on 7/8/11
- Samples: PC1, PC2, GFP+, GFP-, E9.5, E11.5, E13.5m, E13.5f
- 75ul sheared DNA store at 0.5ml non-stick tube over the weekend
- Use NEB master mix kit for Library prep
End repair[edit]
Reaction sheared DNA 75ul Master mix buffer 10ul Enzyme mix 5ul H2O 10ul Total 100ul 20C for 30min
Purification with AMPure beads # 150ul beads to 100ul reaction (kapa lib prep kit suggested), mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 30ul DNA from beads
After end | ng/ul | ng |
GFP+ | 0.35 | 10.5 |
GFP- | 0.24 | 7.2 |
E9.5 | 0.39 | 11.7 |
E11.5 | 1.29 | 38.7 |
E13.5m | 0.54 | 16.2 |
E13.5f | 2.27 | 68.1 |
A tailing[edit]
Reaction End-repaired DNA 30ul Master mix buffer 5ul Enzyme mix 3ul H2O 12ul Total 50ul 37C for 30min
Purification with AMPure beads # 90ul beads to 50ul reaction, mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 20ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 18ul DNA from beads, 1ul left in tube for Qubit measurement
Adapter ligation[edit]
- 30uM Y-adapter made by Alice few weeks ago
- 0.08ul for sample 1,3,4,5,7 and 0.4ul for 2,6,8
- 50ul total with 10ul buffer and 5ul Quick T4 ligase; RT for 30min
' | ng/ul | ng | ug | pmole | adaptor | ul (30uM) |
PC2 | 0.91 | 16.4 | 0.016 | 0.12 | 2.42 | 0.08 |
GFP+ | 0.29 | 5.27 | 0.005 | 0.04 | 0.76 | 0.03 |
GFP- | 0.23 | 4.14 | 0.004 | 0.03 | 0.6 | 0.02 |
E9.5 | 0.4 | 7.22 | 0.007 | 0.05 | 1.06 | 0.04 |
E11.5 | 1.61 | 28.98 | 0.029 | 0.22 | 4.4 | 0.15 |
E13.5m | 0.48 | 8.59 | 0.009 | 0.07 | 1.36 | 0.05 |
E13.5f | 2.72 | 48.96 | 0.049 | 0.37 | 7.42 | 0.25 |
Purification with AMPure beads # 50ul beads to 50ul reaction, mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 22ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 20ul DNA from beads, 1ul left in tube for Qubit measurement
PCR amplification[edit]
Sample | ng/ul | ng |
PC1 | 0.15 | 2.7 |
PC2 | 0.87 | 15.61 |
GFP+ | 0.25 | 4.52 |
GFP- | 0.24 | 4.3 |
E9.5 | 0.31 | 5.51 |
E11.5 | 1.22 | 21.96 |
E13.5m | 0.51 | 9.22 |
E13.5f | 2.63 | 47.34 |
Pre-run[edit]
- Pre-run with PC1 (lowest amount of template) and PC2 (relatively high amount of template)
- 50ul total: 25ul Phusion master mix, 1ul 10uM PCR primer each
- 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (15 cycles); 72C 5min
File:7.11.11 pre-run gel check.jpg
Something wrong with the loading buffer, use a new one!
Sample run[edit]
Sample | ng/ul | ul | ng |
PC1 | 0.15 | 18 | 2.7 |
PC2 | 0.87 | 18 | 15.61 |
GFP+ | 0.25 | 10 | 2.5 |
GFP- | 0.24 | 10 | 2.4 |
E9.5 | 0.31 | 10 | 3.1 |
E11.5 | 1.22 | 2 | 2.4 |
E13.5m | 0.51 | 5 | 2.5 |
E13.5f | 2.63 | 1 | 2.6 |
- 50ul total: 25ul Phusion master mix; 1ul 10uM Pf; 1ul 10uM Pr.ID1 to 6
- 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (12 cycles); 72C 5min
QPCR curve
Size selection[edit]
Continue with lab note on 7.12.11 [1]