Electrotransformation of E. Coli

From 2010.igem.org

1.Chill electroporation cuvettes on ice

2.Prepare the SOC media by adding MgCl2 and Glucose solutions to prepare SOB media and pipette 900 ul into the desired amount of recovery vials

3.Remove # vials containing 100 ul electro-competent cells from the -80 degrees Celsius freezer.

     a.Thaw vials on ice

4.Turn on electroporator and set voltage to 1.5 kV

5.Add 5 ul of ligated DNA sample to 100 ul thawed electrocompetent cells on ice.

     a.Swirl tip around gently in cells to mix DNA and cells.

6.Place cells back on ice to ensure they remain cold.

7.Pipette 100 ul of cell-DNA mixture to cuvette.

8.Wipe off excess moisture from outside of cuvette.

9.Place cuvette in chamber of electroporator.

10.Pulse the cells by pressing button on electroporator twice.

11.Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to the labeled recovery tube.

12.Let cells recover at room temperature for 1-2 hours.

13.Plate 100 ul of electroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic.

     a.Incubate plate overnight at 37 degrees

14. Leave remaining SOB-cell mixture on the bench-top overnight.