AlanFung:LabNotes/Sequencing/2009-12-1
Jump to navigation
Jump to search
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 | 4 |
DNA after USER Digestion | 33 |
S1 Nuclease (10U/ul) | 1 |
ddH2O | 2 |
- 37C 10mins
- Minelute purification. Elute w/16uH2O