Team:Arizona State/Lab/Protocols/Transformation

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Protocols: Transformation


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NEB (10-beta and Turbo)


  1. Thaw a tube of NEB Turbo/NEB 10-beta Competent E.coli cells until the last ice crystals disappear. Mix gently and carefully pipette 50 uL of cells into a transformation tube on ice.
  2. Add 1-5 uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42 C for exactly 30 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 950 uL of room temperature SOC into the mixture.
  7. Place at 37 C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plate to 37 C.
  9. Spread 50-100 uL of each well-mixed dilution onto a selection plate and incubate 8-12 hours to overnight at 37 C. Alternatively, incubate at 30 C for 16 hours or 25 C for 24 hours.


TSS


  1. Thaw 50ul prepared competent TSS cells on ice.
  2. Add approximately 1 ul prepped plasmid to the thawed cells. Make sure the culture is thoroughly mixed.
  3. Incubate for 30 minutes on ice.
  4. Incubate for 30 seconds at 42C (in a water bath).
  5. Incubate for 2 minutes on ice.
  6. Add 1 ml room temperature SOC.
  7. Incubate for 1 hour at 37C with shaking.
  8. Plate with appropriate antibiotic.
  9. Grow overnight at 37C.