AlanFung:LabNotes/Sequencing/2009-11-24

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Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set[edit]

  • 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[edit]

A-D A-D X2 A-B X2
H2O 41.46 82.91 41.64 83.28
Amp F6.3NH2(100uM) 0.2 0.4 0.2 0.4
Amp R6.3NH2 (100uM) 0.2 0.4 0.2 0.4
dUTP (1mM) 2 4 2 4
50X SYBG I 0.4 0.8 0.4 0.8
2X Master Mix 50 100 50 100
Template 5.75 11.49 5.56 11.12
Total 100 200 100 200


PCR program: 94 °C for 3 min, 5 cycles of 94 °C for 45 sec, 55 °C for 45 sec, 72 °C for 45 sec, and finally 72 °C for 3 min.

  • Qiaquick purification, elute with 32ul

USER Digestion[edit]

DNA 30
USER 3
Total 33
  • 37C 2 hours

S1 Nuclease Digestion[edit]

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