AlanFung:LabNotes/Sequencing/2009-12-1: Difference between revisions
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>Alan6017518 (New page: ==Construction of CV-iB libraries with the USER/S1 protocol== ===PCR with dUTP=== *Mix CV-iB #A and #C at 3:1 ratio. 10ul A + 1ul C. *Mix CV-iB #B, #D, #E at 2:1:1 ratio. 4ul B + 4ul D +...) |
>Alan6017518 |
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Line 22: | Line 22: | ||
| S1 Digestion|| | | S1 Digestion|| | ||
|- | |- | ||
| 10X S1 Nuclease Buffer|| | | 10X S1 Nuclease Buffer||8 | ||
|- | |- | ||
| DNA after USER Digestion|| | | DNA after USER Digestion||66 | ||
|- | |- | ||
| S1 Nuclease (10U/ul)|| | | S1 Nuclease (10U/ul)||2 | ||
|- | |- | ||
| ddH2O|| | | ddH2O||4 | ||
|- | |- | ||
| | | | ||
Line 35: | Line 35: | ||
*37C 10mins | *37C 10mins | ||
*Minelute purification. Elute w/16uH2O | *Minelute purification. Elute w/16uH2O | ||
===End repair=== | ===End repair=== | ||
===A-tailing reaction=== | ===A-tailing reaction=== |
Revision as of 17:18, 1 December 2009
Construction of CV-iB libraries with the USER/S1 protocol
PCR with dUTP
- Mix CV-iB #A and #C at 3:1 ratio. 10ul A + 1ul C.
- Mix CV-iB #B, #D, #E at 2:1:1 ratio. 4ul B + 4ul D + 3ul E.
x2 DNA 10ul 2X Taq Master mix: 200ul 1mM dUTP: 8ul 100uM AmpF6.3NH2: 0.8ul 100uM AmpR6.3NH2: 0.8ul H2O 181ul 94C 2min -> 8x (94C 30sec -> 60C 30sec -> 72C 30sec) -> 72C 3min. Purify each amplicon with two Qiaquick columns. Elute w/ 32ul EB buffer in each tube.
USER digestion
DNA 60ul USER 6ul 37C 1 hour
S1 Nuclease digestion
S1 Digestion | |
10X S1 Nuclease Buffer | 8 |
DNA after USER Digestion | 66 |
S1 Nuclease (10U/ul) | 2 |
ddH2O | 4 |
- 37C 10mins
- Minelute purification. Elute w/16uH2O