User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/03/27

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Before Starting

    • Equilibrate a water bath at 42 degrees C
    • Warm the vial of SOC medium to room temperature
    • Warm the plates at 37 degrees C in incubator for 30 minutes

Cell Transformation

The objective of this session was to insert plasmid DNA into cells in order to transform the cells. The protocol from New England Biolabs for transformation (C2527) was followed.

Protocol

  1. Thaw on ice, one 50 uL vial of One Shot(R) cells for each ligation/transformation
  2. A stock solution containing the plasmid DNA was made
    1. 8.9ug of DNA were diluted into 890uL of sterilized water
    2. This solution along with the tube in which the mixture will occur were also placed on ice pending the thawing of the cells.
  3. Pipet 1 to 5uL of each ligation reaction directly into the competent cells and mix by taping gently. Do not mix by pipetting up and down. Store the remaining ligation reaction at -20 degrees C
    1. 2uL of the ligation mixture was added to the cells
  4. Incubate the vials on ice for 30 minutes
  5. Incubate for exactly 30 seconds in the 42 degree C water bath. Do not mix or shake.
  6. Remove vial from water bath and place on ice.
  7. Add 250uL of pre-warmed SOC medium to each vial (SOC iix a rich medium sterile technique must be practiced to avoid contamination.)
  8. Place the vial in a micro centrifuge rach on its side and secure with tape to avoid losing vial. Shake the vial at 37 degrees C for exactly 1 hour at 225rpm in a shaking incubator.
  9. Spread 20 to 200uL from each transformation vial on separate, labeled LB agar plates. WE recomment you plate two different volumes. NB: You may have to dilute cells 1:10 to obtain well-spaced colonies.
  10. Store the remaining transformation reaction at +4 degrees C and plate out the next day if desired.
  11. Invert plates and incubate at 37 degrees C overnight
  12. Select colonies and analyze plasmid isolation, PCR, or sequencing



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