User:Meng Xiao He/Notebook/fall08/2008/11/02

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Ligations/transformations

  • XmaI heat killed
  • vectors treated with ant. phosphatase
  • phosphatase killed
  • fragments column purified
  • used NEB T4 ligase b/c quick ligases have PEG

DH5a, Kan+Xgal:

  • TOPO + each flank's original PCR
  • pUC19 + each flank
  • pUC19 neg control
  • 4ul per transformation


pir+ electrocomp cells, LB spread w/ Gm+Xgal:

  • each pER21 prep + each flank
  • pER21 neg control

Rx

  • 8ul DNA mix (2:6), 1ul ligase, 1ul buffer
  • 20 min ligation



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