IGEM:Virginia 2012/Protocols/Phage Amplification

Overview

Below is the procedure for producing a new phage suspension from bacteria that has the phage genome integrated.

Materials

Part I

• Agar plate (+ chloramphenicol)
• Plastic loop

Part II

• 14 mL Falcon tube
• Liquid LB medium
• Plastic loop
• 10μg/μl Streptomycin
• Pipettes with filter tips
• 1 L Erlenmeyer Flask
• Foil
• 2 cuvettes
• 0.5μg/μl Mitomycin C

Part III

• Chloroform
• Conical tube
• 20% PEG/ 2.5M NaCl
• SM buffer

Procedure

Part I. Streaking infected B. bronchiseptica plates and incubating.

1. Get a plate with the appropriate antibiotic from the cold room. Label the plate with the name of the bacteria, type of media, the date, and your initials.
2. Get the freezer key, and go to the -80 °C freezer. Remove the bacteria which is infected with phage from a box labeled “Frozen Stock”.
3. Working in the hood opposite the freezer, streak cells onto the plate using the loop.
4. Return to the lab. Wrap the plates with parafilm and put them in the 37°C incubator until colonies appear (several hours or overnight).

Part II. Preparing the liquid culture.

1. Add 3 mL of liquid LB medium to the 14 mL Falcon tube.
2. Use the loop to transfer bacteria from the plate to the Falcon tube. Stir the liquid medium gently to dislodge the bacteria and then discard the used loop in the biohazard waste bin.
3. Add 6 μl of streptomycin to the Falcon tube.
4. Place the Falcon tube in the incubator for 2 hours at 37°C.
5. Place the plate in the 4°C freezer or cold room. Plates can be stored in the cold room for up to ~2 weeks before the cells go bad. When the plates are no longer needed, discard them in the biohazard waste bin.
6. Turn on the spectrophotometer (it must be turned on at least 15 minutes prior to use). Set absorbance to 600 nm and setting to ‘basic’.
7. Wait for two hours
8. Label an Erlenmeyer flask with the name of the bacteria, the medium, the date, and your initials.
9. Remove the Falcon tube from the incubator.
10. Using a motorized pipette filler, add 20 mL of liquid LB media to the Erlenmeyer flask
11. Using a pipette, add 40μl of streptomycin.
12. Pipette 2 mL of the culture from the Falcon tube into the Erlenmeyer flask.
13. Cover the Erlenmeyer flask with foil and place it in the incubator for 1 to 1.5 hours at 37°C.
14. Remove the Erlenmeyer flask from the incubator and label the cuvettes.
15. Pipette 1 mL of liquid LB medium into one cuvette.
16. Pipette 1 mL from the Erlenmeyer flask into the second cuvette. Return the Erlenmeyer flask to the incubator.
17. Take the cuvettes to the spectrophotometer. Set the absorbance measurement for the LB cuvette to zero by pressing ‘blank’. Then take the measurement for the cuvette containing the culture.
18. If the O.D. (optical density) is less than 0.6 for the culture, continue incubating the flask until the O.D is around 0.6. When the O.D. is around 0.6, continue with the procedure below.
19. Get the Erlenmeyer flask from the incubator.
20. Using the C₁V₁=C₂V₂ formula, determine how much mitomycin C to add to the Erlenmeyer flask so that the final concentration is 2μg/ml. Pipette that amount of mitomycin C into the flask.
21. Continue incubating the Erlenmeyer flask for 3 hours at 37°C.

Part III. Isolating the phage.

1. Get the Erlenmeyer flask from the incubator.
2. To find the amount of chloroform to add, divide the volume of the culture in the Erlenmeyer flask by 25. Pipette this amount of chloroform into the Erlenmeyer flask.
3. Return the Erlenmeyer flask to the incubator for a few minutes.
4. Pour culture in Erlenmeyer flask into a conical tube.
5. Put the conical tube in the centrifuge and spin it down at 12krpm for 15 minutes.
6. Transfer to a new tube.
7. Add an equal volume of PEG/NaCl (probably 20 mL) to the conical tube.
8. Vortex the tube.
9. Put the conical tube in the 4°C freezer and store overnight (or over the weekend).
10. Take the conical tube to the centrifuge and spin it down at 12k rpm for 15 minutes.
11. Discard supernatant in the sink. Let tube sit upside-down for 10-15 minutes to dry. Resuspend the pellet in 500 μL of SM buffer. Transfer to a 1.5 mL tube.
12. Spin down, and collect the supernatant (10 krpm, 10 min)
13. Prepare a 3 mL liquid culture using the plate that was already streaked and the procedure for Part II.
14. Place Falcon tube in oscillating incubator for 5-6 hours
15. Perform a plaque assay to determine the phage titer.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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