Keating:Transformation

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Transformation

Transformation protocol (for pure plasmid)

  1. Do in 1.5mL eppendorf tubes
  2. 50-100ul competent cells (XL1-Blue or BL-21 that were thawed on ice) + 2ul DNA
  3. Mix by inverting
  4. Place on ice for 10 minutes
  5. Place in heat block at 42 degrees C for 2 minutes
  6. Place on ice for 2 minutes
  7. Add 700ul of LB
  8. Incubate at 37 degrees C for 40 minutes
  9. Plate about 100ul of cells on antibiotic plate
  10. Incubate at 37 degrees C overnight

Transformation after ligation reactions

  1. Use ~150ul compentent XL1-Blue
  2. Place on ice for 10 minutes
  3. Place at 42 degrees C for 2 minutes
  4. Place on ice for 2 minutes
  5. Add 700ul of LB
  6. Place at 37 degrees C for 40 minutes
  7. Spin down cells at 3,000-5,000 rpm
  8. Pipet off about 500ul
  9. Resuspend pellet in remainder.
  10. Plate ½ of the remainder (175ul)
  11. Save the other half in the fridge.
  12. If the plate doesn't have any colonies the following day, you can try plating the other half.