CC6: Transformation into Top10 using chemical competence

Protocol

1. Switch on water bath to 42 degrees.

2. Aliquot out LB media and place in water bath (roughly 1 ml for each sample)

3. Chill DNA and 15ml round bottom tubes on ice (1 for each sample)

4. When all this is done, take out cells from -80 degrees freezer and thaw on ice.

5. When cells are thawed, add 30ul of cells to each round bottom tube.

6. Add 3ul of registry DNA or 1ul of midi DNA to tube. Swirl gently.

7. Incubate on ice for 30 min.

8. Place in 42 degrees water bath for 30 sec. (using timer- this duration is critical)

9. Incubate on ice for 2 min.

10. Put in a 37 degrees incubator with shaking for 1 hour. (still set a timer for this step)

11. Centrifuge cells and resuspend them in a much smaller volume (slightly more than 100ul).

12. Add 100ul of cell solution onto agar plates with appropriate resistance. Spread evenly.

13. Leave out in open to dry.

14. After the plates have dried, put in 37 degrees incubator overnight.