Team:Waterloo/Notebook/Protocols/Transformation

 Transformation 

Before you start  Cells lose their competence once thawed, so be sure to: a) keep them on ice at all times. b) use cells immediately after thawing. c) discard any unused thawed cells.

Materials  Competent cells DNA Ice + Ice bucket 1.5mL microfuge tube Glass Spreader Ethanol Agar Plate with desired antibiotic resistance Alcohol burner

Instructions 1. Thaw competent cells on ice. Include 2 extra ones for a Negative (no DNA) and Positive control. 2. In a 1.5 mL microfuge tube, add 50 μL of cells and 1 μL of DNA, and pipette up and down to mix. 3. Incubate on ice for 30 min. 4.  Transfer directly to 42C heat block for 45 s to “heat shock”. 5. Transfer to ice for 2 min. 6.  Add 500 μL of LB. 7.  Incubate at 37C for 1 h on the shaker. 8. Centrifuge tubes at 13,000rpm for 2 mins. 9. Decant most of the supernatant. Leave approximately 50 μL. 10. Prepare and label plates with date and construct names. Let the plates dry in the flow hood if necessary.

For each sample, the following steps must be carried out quickly in the laminar flow hood to ensure proper spreading of cells. 11. In the laminar flow hood, resuspend pellet in remaining liquid by pipetting up and down. 12. Transfer to center of plate. 13. Dip glass spreader in ethanol and flame. 14. Let the glass spreader cool by gently touching the agar. 15. Spread liquid around plate until it appears dry. 16. Seal plates with Parafilm or put in a bag. 17. Incubate inverted in 37 degrees Celsius incubator overnight.