AlanFung:LabNotes/Sequencing/2009-12-1

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Revision as of 17:18, 1 December 2009 by >Alan6017518 (→‎S1 Nuclease digestion)
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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

End repair

A-tailing reaction

Ligation

Size selection

PCR