Team:UCAS-China/People/Notebook

Notebook can be downloaded here

Preparation of competent cells

1. Streak competent cells on LB plates, and incubate overnight.

2. Pick a single colony and incubate at 37°C in 4ml LB medium.

3. Add 100l of bacteria solution to 100ml LB medium in conical flask.

4. Shake the flask at 37 °C until the OD600 reaches 0.4-0.5, about 3h.

5. Place the conical flask quickly on ice, violently oscillate it to cool it down quickly.

6. Transfer the medium to a pre-cooled centrifuge tube, centrifuge at 4500 rpm for 10 min, and discard the supernatant.

7. Add 2/3 volume of pre-cooled CaCl2-MgCl2 mixture to each tube, resuspend the cells, and ice bath for 10 min.

8. 4 degrees, centrifuge at 4500rpm for 10min, completely discard the supernatant

9. Add 1/25 volume of pre-cooled 0.1M CaCl2 solution to each tube, resuspend the cells, and ice bath for 10 min.

10. Add 7% volume of pre-cooled DMSO to each tube, ice bath for 10 min, dispense into 1.5 ml EP tube, dispense 100 per EP tube, and store in -80 refrigerator.

Bacteria strain preservation

1. Mix 900 bacteria solution with 900 glycerin (60%)

2. Store the mixture in a -80 degree refrigerator