Brown: Team Resistance/12 October 2008

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.
 * 3:35 PM: 4 microliters of ligation product added to competent cells and left in ice for 20 minutes.
 * 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath or thermocycler for 60 seconds.
 * 1) Both tubes are placed back in the ice for two minutes.
 * 2) Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
 * 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
 * 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)
 * 1) Plates are incubated upside down for 24 hours in 37 degrees C.