Brown: Team Resistance/12 October 2008


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12 October 2008

  • Transformation of our ligation of the S105 gene into the Standard Biobrick Vector with DH5alpha competent cells.

Transformation Procedure

  1. Place both in ice. The control in this experiment is a negative control because no DNA is added to the competent cells.
  2. Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used.
  3. 3:35 PM: 4 microliters of ligation product added to competent cells and left in ice for 20 minutes.
  4. 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath or thermocycler for 60 seconds.
  5. Both tubes are placed back in the ice for two minutes.
  6. Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
  7. 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
  8. 5:20 PM: Three solutions are plated on LB ampicillin resistant plates. Spreader dipped in ethanol and passed through a flame to ensure sterilization before plating. The three plates contained: Plate 1) 100 microliters of the control Plate 2) 100 microliters of LB/cells (1:1 ratio) Plate 3) Dilution of 99 microliters water and 1 microliter of LB/cells (100:1 ratio)
  9. Plates are incubated upside down for 24 hours in 37 degrees C.