Protocols
Gradient plates
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”. All materials should be kept as sterile as possible.
- Draw a central cross on the lid of a Petri dish
- 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.
- 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.
- 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.
- 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.
- 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.
Chemically-Induced Competence
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
- Introduce cells to culture tube containing 5 mL LB medium
- Shake overnight at 37°C
- Slate 200 µL of culture on separate LB plates
- Incubate overnight at 37°C
- Add 1.5 mL of 50 µM CaCl2 into microcentrifuge tube
- Cool tube on ice for a minimum of 10 minutes
- Scrape colonies off plate until 1x0.5 cm smear is achieved
- Swirl scraper in CaCl2 until all cells removed, then vortex tube
Liquid Nitrogen
- Introduce cells to two culture tubes, each with 5 mL LB medium
- Shake overnight at 37°C
- Add both cultures to a flask containing 250 mL LB medium
- Shake at 37°C for 3-4 hours, checking periodically until 0.55 ± 0.05 at OD600 is reached
- Place flask on ice for 10 minutes, then transfer to centrifuge bottles
- Spin at 2500x g for 10 minutes at 4°C
- Decant supernatant, then add 80 mL ice-cold Inoue transformation buffer
- Resuspend cells by swirling slowly, not vortexing
- Spin at 2500x g for 10 minutes at 4°C
- While spinning, chill one hundred microcentrifuge tubes at -80°C
- Decant supernatant, resuspend in 20 mL ice-cold Inoue transformation buffer
- Mix DMSO (7% of final volume in bottle), then store on ice for 10 minutes
- Transfer 200 µL to each pre-chilled tube
- Dip tubes in liquid nitrogen for 5 seconds, then store at -80°C
Plasmid Purification
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.
- Prepare overnight cultures of E. coli in 5 mL LB medium (optional: chemical selection)
- Transfer culture to microcentrifuge tube
- Spin for 1 minute at 14,000 rpm at room temperature, then decant supernatant
- Transfer culture, spin, and decant again, if needed
- Resuspend bacterial pellet in 250 µL buffer P1 (which contains RNase A)
- Add 250 µL P2 buffer and invert 4-6 times, not vortex
- Add 350 µL N3 buffer and invert 4-6 times immediately
- Centrifuge for 10 minutes
- Pipette supernatant into spin column attached to a vacuum apparatus
- Turn on vacuum
- Wash spin column with 500 µL of PB buffer
- After PB buffer is removed, wash spin column with 750 µL of PE buffer
- Remove spin column from vacuum after buffer is fully removed, and attach it to column bottom
- Centrifuge for 1 minute to remove residual buffer
- Discard column bottom, and attach spin column to 1.5 mL microcentrifuge tube
- Add 50 µL of EB/TE or any low salt buffer to center of spin column to elute DNA
- After resting for 1 minute, centrifuge for 1 minute
- Use a spectrophotometer to measure purity and concentration for storage and for further experiments
Overnight Cultures
- Add 5 mL of sterile LB medium to autoclaved 20 mL culture tube
- Mix following antibiotic concentration, if needed
- kanamycin= 1 µL/mL
- chloramphenicol= 0.6 µL/mL
- ampicillin= 1 µL/mL
- tetracycline= 1 µL/mL
- Carefully, pick a colony with a sterile pipette tip and eject tip into culture tube
- Incubate in shaker at 37°C for a minimum of 12 hours
LB Agar Plates
- Add 25 g LB powder mixture to 1 L flask, and fill with 800 mL de-ionized water
- Add stir bar to flask and mix on stir plate
- Ensure that stir bar is centered in flask, and maintain watch, as flask may fall
- After mixture is fully dissolved in water (~10 minutes), add de-ionized water until final volume is 1 L
- Add 3.75 g agar into four 500 mL autoclave-safe bottles, and pour 250 mL of the LB mixture into each bottle
- Autoclave bottles, and ensure that bottle caps are screwed just loose enough to lift the bottle by grabbing the cap
- This will prevent the bottles from exploding from internal pressure, and will also prevent the formation of a vacuum, which can cause contamination
- After autoclaving, leave the caps loose until the agar is comfortable to touch
- Antibiotics and other chemicals may be mixed in at this stage
- kanamycin= 1 µL/mL
- chloramphenicol= 0.6 µL/mL
- ampicillin= 1 µL/mL
- tetracycline= 1 µL/mL
- Pour 25 mL of molten agar into each sterile petri dish
- Let petri dishes cool down until agar solidifies, then it can be used immediately, or stored in fridge
- If stored, let agar warm to room temperature before plating, so bacteria are not shocked from rapid temperature changes, leading to less colonies
PCR (50 µl reaction)
- Create a working stock (5 ng/µL) of template DNA, and add 1 µl to PCR tube
- Add 2.5 µl each of 10 µM forward and reverse primers to PCR tube
- Add 1 µl of 25 µM dNTPs
- Add 10 µl of 5X HF Phusion buffer, along with 0.5 µL Phusion enzyme
- Adjust final tube volume to 50 µl with highly purified water
- Try to minimize bubble formation, as this can hinder Phusion from functioning optimally
- Set the PCR machine with appropriate denature, annealing, and extension temperatures, as well as appropriate durations and cycles
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