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Transformation

• written by Christy

Transformation protocol (for pure plasmid)

1. Do in 1.5mL eppendorf tubes
2. 50-100ul competent cells (XL1-Blue or BL-21 that were thawed on ice) + 2ul DNA
3. Mix by inverting
4. Place on ice for 10 minutes
5. Place in heat block at 42 degrees C for 2 minutes
6. Place on ice for 2 minutes
7. Add 700ul of LB
8. Incubate at 37 degrees C for 40 minutes
9. Plate about 100ul of cells on antibiotic plate
10. Incubate at 37 degrees C overnight

Transformation after ligation reactions

1. Use ~150ul compentent XL1-Blue
2. Place on ice for 10 minutes
3. Place at 42 degrees C for 2 minutes
4. Place on ice for 2 minutes
5. Add 700ul of LB
6. Place at 37 degrees C for 40 minutes
7. Spin down cells at 3,000-5,000 rpm
8. Pipet off about 500ul
9. Resuspend pellet in remainder.
10. Plate ½ of the remainder (175ul)
11. Save the other half in the fridge.
12. If the plate doesn't have any colonies the following day, you can try plating the other half.