AlanFung:LabNotes/Sequencing/2009-11-24: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 1: Line 1:
==Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set==
==Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set==
*To make 100ng of CVF-N set (A-D)
*To make 100ng of CVF-N set (A-D)
*
*A-22.5ng/ul*1.5ul=33.75ng
*B-12.8ng/ul*1.32ul=16.875ng
*C-6ng/ul*4.11=24.64ng
*D-5.4ng/ul*4.56=24.64ng
*Total=100ng
 
 
*To make 100ng of CVF-N set (A-B)
*A-22.5ng/ul*2.96ul=66.6ng
*B-12.8ng/ul*2.60ul=33.3ng
*Total=100ng


==PCR with dUTP and 6.3NH2 Primer==
==PCR with dUTP and 6.3NH2 Primer==

Revision as of 20:29, 24 November 2009

Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set

  • To make 100ng of CVF-N set (A-D)
  • A-22.5ng/ul*1.5ul=33.75ng
  • B-12.8ng/ul*1.32ul=16.875ng
  • C-6ng/ul*4.11=24.64ng
  • D-5.4ng/ul*4.56=24.64ng
  • Total=100ng


  • To make 100ng of CVF-N set (A-B)
  • A-22.5ng/ul*2.96ul=66.6ng
  • B-12.8ng/ul*2.60ul=33.3ng
  • Total=100ng

PCR with dUTP and 6.3NH2 Primer

1 well
H2O 46.2
Amp F6.3NH2(100uM) 0.2
Amp R6.3NH2 (100uM) 0.2
dUTP (1mM) 2
50X SYBG I 0.4
2X Master Mix 50
Template 1
Total 100
  • Qiaquick purification, elute with 32ul

USER Digestion

DNA 30
USER 3
Total 33

S1 Nuclease Digestion

S1 Digestion
10X S1 Nuclease Buffer 4
DNA after USER Digestion 33
S1 Nuclease (10U/ul) 1
ddH2O 2
  • 37C 10mins
  • Minelute purification. Elute w/16uH2O

End Repair