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

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Line 27: Line 27:
  Enzyme mix            3ul
  Enzyme mix            3ul
  H2O                  12ul
  H2O                  12ul
  '''Total               50ul'''
  '''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 meansurement
  # 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

Adapter ligation

PCR amplification

Size selection