Team:Alberta/Protocols

The following procedure is to be used to find valuesrelated to diffusion. All time values should be converted to seconds, from whenthe antibiotic was plated, and all distance values should be recorded incentimeters, from the edge of the well to the first sign of life at the edge ofthe “kill zone”. All materials should be kept as sterile as possible, barringthe damage of cells that are being plated.

1. Draw a central cross on the cap of a Petri dish of radius 4.6cm
2. Sterilize a tall cylindrical magnet of a height near, but not at the depth of the plate (a well that reaches the bottom of the plate will allow the antibiotic in question to seep under the agar rather than diffuse through it) and radius 0.25cm. Place the sterilized magnet on the inside of the Petri dish cap and, from outside the cap, adjust and secure the sterilized magnet with another magnet.
3. Melt and pipette 25mL of LB agar into the Petri dish, and place the cap on top, allowing the magnet to rest in the molten agar. Let rest until solid and cool, then remove the cap and allow the surface to dry until it is free of excess moisture.
   Steps 4 and 5 may be completed in any desired order, depending on the approximate amount of time the substanceis meant to diffuse for.
   
4. Pipette 50µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill any. Immediately place the plate in a 37ºC incubator.
5. Evenly plate 200µL of cells with a resistance to the antibiotic over the flat surface of the plate. Immediately place the plate in a 37ºC incubator.
6. Begin watching for results within 2 hours of plating. These will come in the form of a slight difference in texture between the zone in which cells are growing, and the zone in which they are not. It will be very subtle, and may need to be observed by shining light through the agar, or placing the plate on a black backdrop. As soon as it is observed, the radius of the zone must be measured from the edge of the well. The zone may expand. Continue recording the size and time until it stops changing.

The protocols described below were used to create competent cells of Top10 and TG-1 Escherichia coli strains. The Calcium Chloride protocol uses less steps, is easier to perform, and produces competent cells faster than the Liquid Nitrogen procedure. However, we found that the competence efficiency was higher using the Liquid Nitrogen protocol.

Calcium chloride

Liquid Nitrogen

1. Introduce cells to two culture tubes, each with 5 mL LB medium
2. Shake overnight at 37°C
3. Add both cultures to a flask containing 250 mL LB medium
4. Shake at 37°C for 3-4 hours, checking periodically until 0.55 ± 0.05 at OD600 is reached
5. Place flask on ice for 10 minutes, then transfer to centrifuge bottles
6. Spin at 2500x g for 10 minutes at 4°C
7. Decant supernatant, then add 80 mL ice-cold Inoue transformation buffer
8. Resuspend cells by swirling slowly, not vortexing
9. Spin at 2500x g for 10 minutes at 4°C
10. While spinning, chill one hundred microcentrifuge tubes at -80°C
11. Decant supernatant, resuspend in 20 mL ice-cold Inoue transformation buffer
12. Mix DMSO (7% of final volume in bottle), then store on ice for 10 minutes
13. Transfer 200 µL to each pre-chilled tube
14. Dip tubes in liquid nitrogen for 5 seconds, then store at -80°C