IGEM:Peking/2007/Count:Preparation of Competent Cells

Count:Preparation of Competent Cells

Pre-check

1. Make sure the apparatus in the Inventory List in perfect state.
2. Make sure ice, liquid N2 and strains available.

Materials

1. Media
   1. LB(Both liquid media and media containing agar. Add certain antibiotic if it is necessary.);
2. Buffers and Solutions
   1. 0.1M CaCl2;
   2. 80% Glycerin.
3. Special Equipments
   1. EP tubes(1.5mL), micropipette tips, centrifugation bottles(polypropylene tubes, 50mL), graduated flask(100mL*2, 10mL*1), plates and test tubes.

Protocol

1. Sterilization
   1. Including all materials in part2.
   2. Caution(Remind): use some special marks to distinguish the sterilized materials from the unsterilized ones.
2. Preparation after sterilization
   1. Chill the CaCl2 to 4 centigrade degree
   2. Decant LB containing agar into the plates.(If antibiotics are necessary, be sure that they are added when the media temperature is below 60 centigrade degree.) After the cultures cooling down, seal the gap with parafilm. Store the cultures at 4 centigrade degree.
3. Streak the prepared strains onto the agar plates prepared in 4.2. Seal the gaps with parafilm, incubate it at 37'C for 10-16 hours.
4. take 1.6 mL overnight culture to 14.4mL LB bottle.
5. 37C shaking 90min.
6. take 16mL culture to 144mL LB
7. 37C shaking 100~120 min to O.D. 6.0=0.45~0.6
8. on ice for 10min
9. 5000rpm 5min at 4C
10. Add origin volume 1/2 0.1M CaCL2,suspend softly.
11. On ice 20~30 min
12. 5000rpm 5min at 4C
13. origin volume 1/20 0.1M CaCl2
14. suspend cell
15. Add glycerol to 15~20%,store at -80C