IGEM:Virginia 2012/Protocols/Phage Isolation: Difference between revisions
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(New page: ==Overview== Below is the procedure for isolating phage from a mixed bacteria/phage sample. ==Materials== *Chloroform *Conical tube *PEG/NaCl *SM buffer ==Procedure== # Get the Erlenm...) |
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*Chloroform | *Chloroform | ||
*Conical tube | *Conical tube | ||
*PEG/NaCl | *20% PEG / 2.5 M NaCl | ||
*SM buffer | *SM buffer | ||
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# Get the Erlenmeyer flask from the incubator. | # 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 | # 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. | # Return the Erlenmeyer flask to the incubator for a few minutes. | ||
# Pour culture in Erlenmeyer flask into a conical tube. | # 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. | # 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. | # Transfer to a new tube. | ||
# Add an equal volume of [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/PEG/NaCl_Preparation PEG/NaCl] (probably 20 mL) to the conical tube. | |||
# Vortex the tube. | |||
# Put the conical tube in the 4°C freezer and store overnight (or over the weekend). | # 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. | # Take the conical tube to the centrifuge and spin it down at 12k rpm for 15 minutes. | ||
# Resuspend the pellet in | # Discard supernatant in the sink. Let tube sit upside-down for 10-15 minutes to dry. Resuspend the pellet in 500 μL of [http://openwetware.org/index.php?title=IGEM:Virginia_2012/Protocols/SM_Buffer_Preparation SM buffer]. Transfer to a 1.5 mL tube. | ||
# Spin down, and collect the supernatant | # 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. | # 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 | # Place Falcon tube in oscillating incubator for 5-6 hours | ||
# Perform a | # Perform a [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Plaque_Assay plaque assay] to determine the phage titer. | ||
==Notes== | ==Notes== | ||
Latest revision as of 13:59, 31 May 2012
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
- 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.
- Transfer to a new tube.
- Add an equal volume of PEG/NaCl (probably 20 mL) to the conical tube.
- Vortex the 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 500 μL of SM buffer. Transfer to a 1.5 mL 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.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
References
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
