NOTEBOOK

Protocol competent cells

Materials:

Sterile equipment; Cell line E. coli TOP 10; LB liquid culture media, solid LB, SOB and SOC;

Antibiotic (40ug / mL - Chloramphenicol); CCMB caps 80 and NEB; Centrifugal; Tubes for centrifugation; pHmeter; Sterile filter; 2 ml tubes; Resistance plasmid; Spectrophotometer OD 600 nm.

Procedure

• 1) Grow cells of E. coli on a 100 mL Erlenmeyer culture medium LB under constant agitation for 16 hours, at 37 ° C after this time measuring time of 0.5 OD.
• 2) Isolate colonies to the protocol of competence procedure.
• 3) Grow cells in SOB medium. 4 ml of cells were incubated in SOB medium (250 ml) was stirred at 23 ° C until OD reaches 0.3 nm.
• 4) After OD desired transfer the culture to centrifuge tubes background. Weigh and balance the tubes.
• 5) Centrifuge at 3000 g at 4 ° C for 10 minutes.
• 6) Discard supernatant into waste container containing chlorine bleach.
• 7) Resuspend cell pellet with cold CCMB80 buffer in a total volume of 80 ml​​
• 8) Incubate the cells resuspended in an ice bath for 20 minutes.
• 9) Centrifuge at 3000 g at 4 ° C for 10 minutes.
• 10) Decant the supernatant into the waste container containing chlorine bleach.
• 11) Resuspend the cell pellet in 10 ml cold CCMB80 buffer.
• 12) Aliquot 200 µl of a mixture SOC and 50 µl of resuspended cells and measuring the OD (600 nm). Using a mixture of 200 µl of SOC medium and 50 ul CCMB80 as white for measurement. Obtaining of 1 O.D 1.5 nm.
• 13) Finally storing the cells at -80 ° C in glycerol. (30 µl glycerol + 170 µl cell culture).