Rui:LabNotes/Collaborations/2011-7-11: Difference between revisions
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>RuiLiu m (→A tailing) |
>RuiLiu m (→A tailing) |
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Line 27: | Line 27: | ||
Enzyme mix 3ul | Enzyme mix 3ul | ||
H2O 12ul | H2O 12ul | ||
'''Total | '''Total 50ul''' | ||
37C for 30min | 37C for 30min | ||
Line 35: | Line 35: | ||
# 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 19ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | # Air-dry for 15min, re-suspend with 19ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | ||
# Transfer 17ul DNA from beads, 1ul left in tube for Qubit | # Transfer 17ul DNA from beads, 1ul left in tube for Qubit measurement | ||
===Adapter ligation=== | ===Adapter ligation=== |
Revision as of 17:35, 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
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 19ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 17ul DNA from beads, 1ul left in tube for Qubit measurement