Paulsson:Electroporation of E.coli

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Adapted from Current Protocols in Molecular Biology

Materials

BioRad MicroPulser (Bench #6)
Chilled electroporation cuvettes
SOC medium
LB plates containing antibiotics

Transform the cells

1. Thaw electrocompetent cells on ice.

2. Add 1µl DNA (5pg to 0.5µg plasmid DNA) to cells. Mix by tapping the tube or by swirling the cells with the pipet tip.

The volume of DNA added to the cells should be kept small. Adding DNA up to one-tenth of the cell volume will decrease the transformation efficiency 2- to 3-fold. Also, since the resistance of the sample should be high, make sure that addition of the DNA to the cells does not increase the total salt concentration in the cuvette by >1 mM.

3. Transfer the DNA and cells into a pre-chilled cuvette, tap slightly to settle the cells to the bottom and remove all air bubbles.

4. Set the MicroPulser to "Ec2" for 0.2cm cuvettes, or "Ec1" for 0.1cm cuvettes. Refer to the manual for more details on optimizing settings.

5. Wipe the ice and water from the cuvette with a Kimwipe. Place the cuvette into the sample chamber. Apply the pulse by pushing the yellow button until the machine beeps.

6. Remove the cuvette. Immediately add 1 ml SOC medium and transfer to a sterile culture tube.

7. Incubate 30 to 60 min with moderate shaking at 37°C (remember, just 30°C for ts-Rep plasmids).

8. Plate aliquots (100 - 200ul) of the transformation culture on LB plates containing antibiotics. If need be, make serial dilutions of the culture to achieve approx. 100 colonies per plate. Incubate overnight at the appropriate temperature.

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