Difference between revisions of "Team:UCAS-China/People/Notebook"
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<p>1. Mix 900 bacteria solution with 900 glycerin (60%)</p> | <p>1. Mix 900 bacteria solution with 900 glycerin (60%)</p> | ||
<p>2. Store the mixture in a -80 degree refrigerator</p> | <p>2. Store the mixture in a -80 degree refrigerator</p> | ||
| + | <h3>Project:</h3> | ||
<p></p> | <p></p> | ||
<p></p> | <p></p> | ||
Revision as of 06:00, 17 October 2018
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
Project:
