User:Tk/Notebook/MF-xfm/2008/04/25

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Transformations

  • Transform 400 ul sample frozen at -80
  • Thaw on ice
  • Add 16 ul transposome (4/24)
  • Add 80 ul 1 M sorbitol
  • Dispense 4x into 1 mm chilled cuvettes
  • Hold on ice 10 min
  • Electroporate at 1.5 KV
  • Immediately add 1 ml 1161 medium
  • Hold on ice 10 min
  • Incubate in the cuvette for 50 minutes at 30C
  • Plate out 330 ul onto 12x 1161 + tet plates

More transposon construction

  • Grow pTT01 plasmid (10 ml overnight culture + 500 ml LB/Kan in a shake flask)
  • Harvest in conical centrifuge tubes after 9 hour growth
  • Discard supernatent, freeze pellet

PCR transposon construction

  • 100 ul PCR reaction
    • 50 ul Phusion m/m
    • 47 ul water
    • 3 ul ME0 primer
    • cycle 98/30s (98/15s 55/15s 68/1:15 min)x36