Rui:LabNotes/Collaborations/2011-7-11: Difference between revisions

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  '''Purification with AMPure beads'''
  '''Purification with AMPure beads'''
  # 180ul beads to 150ul reaction (kapa lib prep kit suggested), mix 10 times, R.T for 8min
  # 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

Revision as of 17:28, 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

Adapter ligation

PCR amplification

Size selection