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Comparative Physiology @ Guelph

XL-1 Blue or BL-21 Transfection, Miniprep & Miniprep XL-1 blue

XL-1 Blue or BL-21 Transfection, Miniprep

  • Competent cells are fragile and should be kept on ice and handled very gently
  • Go to –80C Freezer and in box 3 get out a (~25ul) frozen aliquot of competent cells
  • Place on ice and wait until it thaws on ice-usually no more than 5 minutes
  • Place a round bottom 2059 tube on ice, add all of thawed cells to bottom of tube
  • Add 1ul BME (1:10 dilution of stock pure beta mercapto ethanol), gently stir
  • Add 1-4ul plasmid DNA, gently stir but do not pipette up and down
  • Leave on ice 5-30 minutes, occasionally gently swirl the tube itself
  • Get a water bath set at 42C
  • Place cells in water bath for 30 sec, no more or no less
  • Immediately place cells on ice for 2 minutes
  • Add 250ul SOC broth, then shake at 37C for 1 hour
  • Pour onto LB agar plate with appropriate antibiotic, spread with sterile loop
  • For BL-21, next day take a loop full of colonies and innoculate 1 liter 2XY T broth

Miniprep XL-1 blue

  • Next day, use a sterilized loop to pick one colony to place in one tube of Lbroth with
  • appropriate antibiotic added, repeat for total 4 colonies
  • Place into 37C shaker overnight
  • Follow Qiagen protocol for mini-prep the next morning