Making Competent DY330 F' ER2738 Cells For Recombination

1. Make 1:50 dilution of O/N in 50ml LB and place in 500ml flask.

2. Grow cells to OD₆₀₀=0.5.

3. Aliquot 10ml cells into sterile 250ml flasks.

4. Place the "induced" flasks in 42°C waterbath and shake for 15 min.

5. Place the "uninduced" flasks in the 30°C shaker for 15 min.

Note: You will have 1 "uninduced" flask as a control and each 10ml "induced" flask can be used for 2 transformations.

6. Place both "uninduced" and "induced" flasks in ice water slurry.

Note: Must use an ice water slurry to cool flasks, not just ice.

7. Swirl flasks to quickly cool cells and let them sit for 15 min.

8. Spin down cells in cold 15ml conical tubes at 3700 rpm for 10min @ 4°C.

9. Decant supernatant.

10. Resuspend in 1ml ice cold sterile water and transfer to Eppendorf tube.

11. Spin @ 4°C at max speed for 20 sec.

12. Aspirate supernatant.

13. Repeat steps 10-12 twice.

14. Resuspend in 200l of ice cold sterile water.

Electroporate Chlor-Sac Cassette with Competent Cells

1. Mix 5l and 20l of cassette with 100l DY330 competent cells.

2. One at a time, pipet each sample into a prechilled (-20°C) electroporation tube.

3. Make sure all of sample is at the bottom of the tube by tapping on the bench top.

4. Place electroporation tube in electroporator with notch side facing towards the inside.

5. Push tube all the way to the end of the electroporator.

6. Set to 2.0kV and tube knob to discharge.

7. Count to 5 (1, 1000, 2, 1000, 3, 1000,etc.)

8. Turn knob to charge.

9. Immediately remove electroporation tube and add 1ml of LB; pipet up and down.

10. Pipet sample back into original Eppendorf tube.

11. Outgrow cells for 1.5hrs, shaking @ 30°C.

12. Plate 100l of cells onto a LB+chlor-tet-20% sucrose plate to check for the SacB gene.

13. For remainder of sample, plate 100l of cells onto chlor-tet plates.

14. Incubate plates @30°C O/N.