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

From ZhangLabWiki
Jump to navigation Jump to search
>RuiLiu
>RuiLiu
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

A tailing

=Adapter ligation

PCR amplification

Size selection