Team:Alberta/Protocols

The following procedure is to be used to find values related to diffusion. All time values should be converted to seconds, from when the antibiotic was plated, and all distance values should be recorded in centimeters, from the edge of the well to the first sign of life at the edge of the “kill zone”. Ensure that all materials are sterile before starting.

1. Draw a central cross on the lid of a Petri dish
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 0.25 cm. Place the sterilized magnet on the inside of the Petri dish lid and, from outside the lid, adjust and secure the sterilized magnet with another magnet.
3. Melt and pipette 25 mL of LB agar into the Petri dish, and place the lid on top, allowing the magnet to rest in the molten agar. Let agar 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 substance is meant to diffuse for.
   
4. Pipette 50 µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill. Immediately place the plate in a 37ºC incubator.
5. Evenly plate 200 µL 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

1. Inoculate cells into culture tube containing 5 mL LB medium
2. Shake overnight at 37°C
3. Plate 200 µL of culture on separate LB plates
4. Incubate overnight at 37°C
5. Add 1.5 mL of 50 µM CaCl₂ into microcentrifuge tube
6. Cool tube on ice for a minimum of 10 minutes
7. Scrape colonies off plate until 1x0.5 cm smear is achieved
8. Swirl scraper in CaCl₂ until all cells removed, then vortex tube

Liquid Nitrogen

1. Inoculate cells into two culture tubes, each with 5 mL LB medium (one culture tube may be used if large enough for aeration)
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

The following protocol is taken from the instructions provided by Qiagen’s QIAprep Spin Miniprep Kit. We changed the rpm of centrifuge from 13,000 to 14,000, and used a vacuum apparatus for select steps, instead of centrifuge.

1. Prepare overnight cultures of E. coli in 5 mL LB medium (optional: chemical selection)
2. Transfer culture to microcentrifuge tube
3. Spin for 1 minute at 14,000 rpm at room temperature, then decant supernatant
4. Transfer culture, spin, and decant again, if needed
5. Resuspend bacterial pellet in 250 µL buffer P1 (which contains RNase A)
6. Add 250 µL P2 buffer and invert 4-6 times, not vortex
7. Add 350 µL N3 buffer and invert 4-6 times immediately
8. Centrifuge for 10 minutes
9. Pipette supernatant into spin column attached to a vacuum apparatus
10. Turn on vacuum
11. Wash spin column with 500 µL of PB buffer
12. After PB buffer is removed, wash spin column with 750 µL of PE buffer
13. Remove spin column from vacuum after buffer is fully removed, and attach it to column bottom
14. Centrifuge for 1 minute to remove residual buffer
15. Discard column bottom, and attach spin column to 1.5 mL microcentrifuge tube
16. Add 50 µL of EB/TE or any low salt buffer to center of spin column to elute DNA
17. After resting for 1 minute, centrifuge for 1 minute
18. Use a spectrophotometer to measure purity and concentration for storage and for further experiments

1. Add 5 mL of sterile LB medium to autoclaved 20 mL culture tube
2. Mix following antibiotic concentration, if needed
   • kanamycin= 1 µL/mL
   • chloramphenicol= 0.6 µL/mL
   • ampicillin= 1 µL/mL
   • tetracycline= 1 µL/mL
3. Carefully, pick a colony with a sterile pipette tip and eject tip into culture tube
4. Incubate in shaker at 37°C for a minimum of 12 hours