AlanFung:Protocol/Construction of Solexa sequencing Library(USER): Difference between revisions
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>Alan6017518 |
>Alan6017518 |
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(5 intermediate revisions by the same user not shown) | |||
Line 4: | Line 4: | ||
| ||1 well | | ||1 well | ||
|- | |- | ||
| H2O|| | | 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)|| | | 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== | |||
{| {{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