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

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==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==
{| {{table}}
{| {{table}}
| ||1 well
| A-D||A-D||X2||||A-B||X2
|-
|-
| H2O||46.2
| H2O||41.46||82.91||||41.64||83.28
|-
|-
| Amp F6.3NH2(100uM)||0.2
| Amp F6.3NH2(100uM)||0.2||0.4||||0.2||0.4
|-
|-
| Amp R6.3NH2 (100uM)||0.2
| Amp R6.3NH2 (100uM)||0.2||0.4||||0.2||0.4
|-
|-
| dUTP (1mM)||2
| dUTP (1mM)||2||4||||2||4
|-
|-
| 50X SYBG I||0.4
| 50X SYBG I||0.4||0.8||||0.4||0.8
|-
|-
| 2X Master Mix||50
| 2X Master Mix||50||100||||50||100
|-
|-
| Template||1
| Template||5.75||11.49||||5.56||11.12
|-
|-
| Total||100
| 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
*Qiaquick purification, elute with 32ul


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|  
|  
|}
|}
*37C 2 hours
==S1 Nuclease Digestion==
==S1 Nuclease Digestion==
{| {{table}}
{| {{table}}
Line 55: Line 69:
*37C 10mins
*37C 10mins
*Minelute purification. Elute w/16uH2O
*Minelute purification. Elute w/16uH2O
==End Repair==

Latest revision as of 21:51, 25 November 2009

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