User:Meng Xiao He/Notebook/fall08/2008/11/19

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One primer Phusion PCR of suicide vectors

  • extension time: 4:30
  • annealing temp: 52C
  • primer: 1ul (in 25ul of rx) of M13F primer from TOPO kit
  • cycles: 50
  • DNA (old minipreps of cbb, cco, Duo): 2ul each
  • for control: TOPO A

Confirmation of pure cultures

  • Phusion PCR new TOPO cco, pER-cbb, pER-cco, pER-Duo#5, 7 colonies of TOPO C plate
  • Rx: 5ul mastermix, 4.3ul water, 0.5ul primer pair, touch of DNA/cells
  • Conditions: 2min initial denaturation at 98, 50s extension, annealing at 57C, 30 cycles

Loss of plasmid

  • Streaked out cultures of WM3064 on LB-DAP-agar, inoculate fresh LB-DAP liquid culture