Team:LIKA-CESAR-Brasil/ProtocolCells

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Revision as of 02:01, 18 October 2014

LIKA | CESAR

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).
Marca LIKA Marca CESAR

Retrieved from "http://2014.igem.org/Team:LIKA-CESAR-Brasil/ProtocolCells"