IGEM:Virginia 2012/Protocols/Phage Isolation
Below is the procedure for isolating phage from a mixed bacteria/phage sample.
- Conical tube
- 20% PEG / 2.5 M NaCl
- SM buffer
- Get the Erlenmeyer flask from the incubator.
- 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.
- Return the Erlenmeyer flask to the incubator for a few minutes.
- Pour culture in Erlenmeyer flask into a conical tube.
- Put the conical tube in the centrifuge and spin it down at 12krpm for 15 minutes.
- Add an equal volume of PEG/NaCl (probably 20 mL) to the conical tube.
- Put the conical tube in the 4°C freezer and store overnight (or over the weekend).
- Take the conical tube to the centrifuge and spin it down at 12k rpm for 15 minutes.
- 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.
- Spin down, and collect the supernatant (10 krpm, 10 min)
- Prepare a 3 mL liquid culture using the plate that was already streaked and the procedure for Part II.
- Place Falcon tube in oscillating incubator for 5-6 hours
- Perform a plaque assay to determine the phage titer.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- Who has experience with this protocol?
or instead, discuss this protocol.