Preparation of competent cells:

Requirements:
24 hour old culture of E.coli in LB broth 200ml LB broth in 1 liter flask 100 mM Cacl2 100 mM Mgcl2 Glycerol 200ml 200ml 10ml (autoclaved) (autoclaved) (autoclaved) (autoclaved)

Material:
a. Tips and pipettes of: 1. 2. 1ml 100l

b. Falcon tubes c. Spectrophotometer d. Ice e. High speed centrifuge machine f. Appendrofs

Procedure:
1. Take 24 hour old culture of E.coli in LB broth 2. Shift 2ml from this to 200ml of LB broth and take its OD value after certain intervals of time in order to adjust it between 0.20-0.30. 3. When the required OD is obtained, immediately shift the flask on ice for exactly 30 minutes to stop the growth of bacteria. 4. After incubation of 30 minutes, pour the culture in falcon tubes (under sterile conditions) and centrifuge at 7500 rpm for 10 minutes. 5. Discard the supernatant and add volume of chilled Mgcl2. Again centrifuge at same speed. 6. Discard supernatant and add volume of chilled Cacl2. Pace the tubes on ice for 20 minutes and then spin at 7500rpm. 7. Discard supernatant and add 1/2 0 volume of cold Cacl2 and 15% cold glycerol while keeping the tubes on ice. 8. Make aliquots of 100l in appendrofs and store at -40 .

How to check the competent cells:

Requirements:
1. 2. 3. 4. Plasmid to be incorporated in competent cells Water bath set at 42 . LB broth L-agar plates containing X-gal, IPTG and ampicillin.

Procedure:
1. 2. 3. 4. 5. 6. 7. Thaw the appendrof containing competent cells at room temperature. Add 1l of plasmid in this vial and place in water bath at 42 for exactly 90 seconds. Shift the vial on ice for 5 minutes. Add 1ml of LB broth in appendrof and incubate at 37 for 3 hours. After incubation of 3 hours, centrifuge the appendrof for 5 minutes at high speed. Discard 900l of medium and resuspend the pellet in remaining 100l medium. Spread 50l of it on LB agar containing X-gal, IPTG and ampicillin and give overnight incubation at 37 . 8. Colonies of blue color will indicate that plasmid has been incorporated successfully.