Team:UQ-Australia/Notebook/Transformation procedure2

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Transformation Procedure 2

1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice.

2. Gently mix cells, then aliquot 100ul competent cells into chilled polypropylene tubes. Add 1.7 ul of B-mercaptoethanol. Gently stir every 2 minutes for 10 minutes.

3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix.

4. Incubate cells on ice for 30 minutes

5. Heat-shock cells for 30 seconds in 42'C waterbath. DO NOT SHAKE

6. Place on ice for 2 minutes

7. Add 900ul of room temperature SOC medium + glucose

8. Shake at 225rpm (37'C) for 1 hour

9. During the 1 hour incubation take agar plates out of fridge and incubate in 37'C incubator for 30 minutes

10. Spread 3 agar plates: 50ul, 100ul, 500ul

11. Incubate at 37'C overnight

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