Team:Newcastle/E. coli Competence

From 2010.igem.org

Revision as of 14:46, 29 July 2010 by Shethharsh08 (Talk | contribs)

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Competent cells preparation

Materials required

  1. Conical flask
  2. 300ml of LB broth
  3. 1/40 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

Procedures

  1. Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/40 volume of an overnight culture of the desired strain.
  2. Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
  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 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
Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon