Rui:LabNotes/Collaborations/2011-7-11: Difference between revisions
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>RuiLiu m (→End repair) |
>RuiLiu m (→End repair) |
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Line 11: | Line 11: | ||
Enzyme mix 5ul | Enzyme mix 5ul | ||
H2O 10ul | H2O 10ul | ||
''' Total 100ul''' | '''Total 100ul''' | ||
20C for 30min | 20C for 30min | ||
Purification with AMPure beads | |||
# 180ul beads to 100ul 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 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min | |||
# Transfer 30ul DNA from beads | |||
===A tailing=== | ===A tailing=== |
Revision as of 17:04, 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 # 180ul beads to 100ul 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 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 30ul DNA from beads