Rui:LabNotes/Collaborations/2011-7-11
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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]