IGEM:Virginia 2012/Protocols/Phage Isolation

Overview

Below is the procedure for isolating phage from a mixed bacteria/phage sample.

Materials

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

Procedure

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. Add an equal volume of PEG/NaCl (probably 20 mL) to the conical tube.
7. Put the conical tube in the 4°C freezer and store overnight (or over the weekend).
8. Take the conical tube to the centrifuge and spin it down at 12k rpm for 15 minutes.
9. Discard supernatant in the sink. Let tube sit upside-down for 10-15 minutes to dry. Resuspend the pellet in 500μl of SM buffer. Transfer to a 1.5ml tube.
10. Spin down, and collect the supernatant (10 krpm, 10 min)
11. Prepare a 3 mL liquid culture using the plate that was already streaked and the procedure for Part II.
12. Place Falcon tube in oscillating incubator for 5-6 hours
13. 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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