Team:Valencia/Transforming Coccus


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Revision as of 02:07, 27 September 2012

Transforming: Synechococcus elongatus PCC7942

Reagents and Materials

  • Synechococcus elongatus PCC7942
  • Plates
  • 0’3 mL BG-11
  • 10 mL of 10mM Nacl
  • Light


  1. Grow 100 ml of S. elongatus.
  2. Harvest 15 ml of cyanobacterial cells by centrifugation for 10 min at 6000gx.
  3. Resuspend the cell pellet in 10 mL of 10mM Nacl and harvest by centrifugation for 10 min at 6000gx.
  4. Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube.
  5. To each 0.3 mL, add between 50 ng and 2 ug of DNA (typically, we use 1-2 ul from a preparation of 100-200ng/ul).
  6. Wrap the tubes in aluminum foil the shield the cells from light and incubate them overnight at 30oC with gentle agitation.
  7. Plate the entire 0.3-mL cell suspension on a BG-11 Medium plate containing the appropiate selective medium.
  8. Incubate the plates at 30oC in constant light for aprox. 5 d until single colonies appear.
  9. Restreak isolated colonies that have the appropiate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.
  10. Grow mutant clones in 100ml of BG-11.