User:Andriana/16 June 2012

From 2012.igem.org

I. Green Experiment Results:

12 hours after plating – about equal, abundant colony growth all across except for treatment D (no colonies):

• Analysis:

• E. coli die even in very low concentration of base.
• No significant difference in colony number among all other plates signifies that ion presence affects E. coli survival when frozen at very low temperatures much more than it does when E. coli are simply suspended in them, but this is not conclusive since the cells were suspended in the ion solutions for only 30 minutes compared to frozen for 24 hours during the red experiment.
• The significant increase in survival rates, despite much lower cell concentration in solutions, also shows that E. coli are, at least to some degree killed by crystal formation during freezing despite the use of a different strain for the green experiment.

• Further Questions:

• Might the origami strain be more resistant to damage by ions?
• By what process do ions increase E. coli death during freezing since this is not apparent during suspension of cells in corresponding ion solutions?
• Could the ampicillin and kanamycin resistance have affected colony #’s in previous (red) experiment? Did the antibiotics in the agar kill survived cells or did the cells with the plasmid die because this difference caused some sort of disadvantage?
• How will the survival and colony number differ if origami cells are frozen for 24 hours at similar conditions but then all plated on plain agar plates?

II. Third (Purple) Experiment

1. Freeze origami (.377 microliters of cells 2 and .256 microliters of cells 4, of E. coli solutions 10x diluted from original stock for approximately 10^3 cells) for 24 hours at -80C in the following solutions:

• A 0.1M NaCl – 4 microliters 5M NaCl to 196 sterile, DIW
• B. 0.01M NaCl - 0.4 microliters 5M NaCl to 199.6 sterile, DIW
• C. 0.005M NaCl - 0.2 microliters 5M NaCl to 199.8 sterile, DIW
• D. 0.01M MgCl - 20 microliters .1M MgCl to 180 sterile, DIW

III. Made more plain agar plates

IV. Autoclaved eppendorf tubes and pipette tips

V. Worked on concentration and dilution calculations for easy primer addition during PCR reactions

VI. Redid failed PCR from previous day