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Team:Valencia/Transforming Coccus
From 2012.igem.org
Transforming: Synechococcus elongatus PCC7942
Reagents and Materials
- Synechococcus elongatus PCC7942
- Plates
- 0’3 mL BG-11
- 10 mL of 10mM Nacl
- Light
Protocol
- Grow 100 ml of S. elongatus.
- Harvest 15 ml of cyanobacterial cells by centrifugation for 10 min at 6000gx.
- Resuspend the cell pellet in 10 mL of 10mM Nacl and harvest by centrifugation for 10 min at 6000gx.
- Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube.
- To each 0.3 mL, add between 50 ng and 2 μg of DNA (typically, we use 1-2 μl from a preparation of 100-200ng/μl).
- Wrap the tubes in aluminum foil the shield the cells from light and incubate them overnight at 30oC with gentle agitation.
- Plate the entire 0.3-mL cell suspension on a BG-11 Medium plate containing the appropiate selective medium.
- Incubate the plates at 30ºC in constant light for aprox. 5 d until single colonies appear.
- Restreak isolated colonies that have the appropiate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.
- Grow mutant clones in 100ml of BG-11.
