Team:Newcastle/E. coli Competence


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Preparing competent E. coli

Materials required

  1. Conical flask
  2. 300ml of LB broth
  3. 1/20 volume of an overnight culture of the desired strain
  4. Ice and ice bucket
  5. eppendorf tubes
  6. TFB1
  7. TFB2
  8. 1.5 ml Microfuge tubes
  9. Ethanol dry ice
  10. -80°C freezer

Preparation of 100 ml of TFB1

Number Chemicals required Volume
1 KAc 30 mM
2 CaCl2 10 mM
3 KCl 100 mM
4 Glycerol 15% (v/v)
5 Distill water 900 ml
6 500 mM MnCl2.4H2O 100 ml

Preparation of 100 ml of TFB2

Number Chemicals required Volume
1 CaCl2 75 mM
2 KCl 10 mM
3 Glycerol 15% (v/v)
4 Distill water 900 ml
5 Na-MOPS pH 7.0 100 mM


  1. Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.
  2. Grow the cell at 37°C, 200 rpm in an orbital incubator.
  3. Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
  4. Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
  5. Repeat the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
  6. Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice .
  7. Store at -80°C.

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