Adapted from protocol given by Joe Graber
Amber Pollack 7/6/05
PREPARATION OF COMPETENT CELLS (E. coli)
The original protocol from Amy uses 1L of E. coli culture. This protocol is scaled down to 100mL, and
makes approximately 25 tubes of 200uL aliquots.
MgCl2∙6H20 FM = 203.3
203.3g/L = 1M
Protocol:
or 20.3g/L=0.1M
Note: STERILIZE EVERTHING (graduated cylinder and pipettes)
1. Grow E. coli in a 3mL LB broth overnight.
need 5.08g/250 mL
2. Place 500uL of O/N culture into 100mL of LB broth.
3. Monitor growth until density reaches 0.45-0.47 OD600.
CaCl2∙2H20 FM=147.0g/L = 1M
14.7g/L = 0.1M
or 3.67g/250 mL
4. Quickly immerse flask in ice, and swirl.
or 1.47g/100 mL
5. Spin out cells at 5000g (5.5 x 103 rpm) for 5 minutes.
//For 100mL, use 2 sterile centrifuge bottles and pre-cooled GSA rotor in floor Sorvall centrifuge//
6. Resuspend cells in 24mL of ice cold MgCl2 (0.1M).
7. Spin out by running centrifuge up to 4000g (5000rpm).
8. Pour off supernatant.
9. Suspend cells in 24mL of ice cold, sterile CaCl2, 0.1M by gentle pipetting
10. Spin again just up to 4000g (5x103 rpm)
11. Pour off supernatant.
12. Suspend in 4.3mL of 0.1M sterile CaCl2, ice cold.
13. Add 700uL of sterile glycerol, mix well.
14. Dispense 200uL to sterile Eppendorf vials. Snap-freeze in dry ice.
15. Set one microfuge tube aside on ice to test for competency and contamination, transforming a portion
of cells with a known concentration of DNA, and a negative control using no DNA.
//Competent cells will uptake the DNA in the first treatment, and plating the recovered culture on selective media will result in
many colonies. In the negative control treatment, no DNA should be present and therefore no antibiotic resistance will be
conferred to the cells—there should be no colonies when this treatment is plated on selective media. The negative control does
NOT guarantee the batch is uncontaminated and it is recommended a no-DNA control be used for all transformations.//