AlanFung:Protocol/Construction of Solexa sequencing Library(USER): Difference between revisions

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>Alan6017518
>Alan6017518
 
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Line 4: Line 4:
| ||1 well
| ||1 well
|-
|-
| H2O||47.4
| H2O||46.6
|-
|-
| Amp F6.3NH2(100uM)||0.2
| Amp F6.3NH2(100uM)||0.2
Line 10: Line 10:
| Amp R6.3NH2 (100uM)||0.2
| Amp R6.3NH2 (100uM)||0.2
|-
|-
| dUTP (1mM)||0.8
| dUTP (1mM)||1.6
|-
|-
| 50X SYBG I||0.4
| 50X SYBG I||0.4
Line 22: Line 22:
|  
|  
|}
|}
*Qiaquick purification, elute with 32ul
==USER Digestion==
==USER Digestion==
{| {{table}}
| DNA||30
|-
| USER||3
|-
| Total||33
|-
|
|}
==S1 Nuclease Digestion==
{| {{table}}
| 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==

Latest revision as of 18:55, 24 November 2009

Sequencing Library Construction with Single Adaptor Protocol[edit]

PCR with dUTP and 6.3NH2 Primer[edit]

1 well
H2O 46.6
Amp F6.3NH2(100uM) 0.2
Amp R6.3NH2 (100uM) 0.2
dUTP (1mM) 1.6
50X SYBG I 0.4
2X Master Mix 50
Template 1
Total 100
  • Qiaquick purification, elute with 32ul

USER Digestion[edit]

DNA 30
USER 3
Total 33

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

End Repair[edit]