Rui:LabNotes/Collaborations/2011-7-11: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''ul (30uM)''' | | 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+||0.29||5.27||0.005||0.04||0.76||0.03 |
Revision as of 22:05, 11 July 2011
Prepare RNAseq library for sequencing
- 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
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
Sample | Concentration | ng/ul |
GFP+ | 0.35 | ug/mL |
GFP- | 0.24 | ug/mL |
E9.5 | 0.39 | ug/mL |
E11.5 | 1.29 | ug/mL |
E13.5m | 0.54 | ug/mL |
E13.5f | 2.27 | ug/mL |
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
- 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
' | 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 23ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 22ul DNA from beads, 1ul left in tube for Qubit measurement