High titre preparation of phage: Difference between revisions
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High titre preparation of phage (phage lysates) can be used for any experiment requiring specific bacteriophages (e.g. for phage typing of strains, generalized transduction or any other experiments). | High titre preparation of phage (phage lysates) can be used for any experiment requiring specific bacteriophages (e.g. for phage typing of strains, generalized transduction or any other experiments). | ||
A single plate (from a [[Streptomyces | A single plate (from a [[Streptomyces Bacteriophage plaque assay]]) generates several millilitres of a high titre (~10<sup>8</sup> pfu/mL) phage lysate. | ||
===Protocol=== | ===Protocol=== | ||
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Instructions: | Instructions: | ||
#Take plates from the [[Streptomyces | #Take plates from the [[Streptomyces Bacteriophage plaque assay]] | ||
#Take the dilution(s) that show almost confluent lysis (You should still be able to distinguish the shapes of individual plaques, but the plate should display as much lysis as possible - to harvest the maximum number of phage. Often it pays to look at the edges of the plates where it looks like there is no growth of the indicator strain – these often display confluent lysis). | #Take the dilution(s) that show almost confluent lysis (You should still be able to distinguish the shapes of individual plaques, but the plate should display as much lysis as possible - to harvest the maximum number of phage. Often it pays to look at the edges of the plates where it looks like there is no growth of the indicator strain – these often display confluent lysis). | ||
#Flood the plates exhibiting almost confluent lysis with 5 ml of Difco Nutrient broth (DNB), and leave at room temperature for 2 hrs. | #Flood the plates exhibiting almost confluent lysis with 5 ml of Difco Nutrient broth (DNB), and leave at room temperature for 2 hrs. | ||
Revision as of 10:35, 5 May 2020
High titre preparation of Streptomyces phage
This protocol has been confirmed to work for the following organisms:
- Streptomyces coelicolor with ΦC31
- Streptomyces venezuelae with ΦSV1
High titre preparation of phage
High titre preparation of phage (phage lysates) can be used for any experiment requiring specific bacteriophages (e.g. for phage typing of strains, generalized transduction or any other experiments).
A single plate (from a Streptomyces Bacteriophage plaque assay) generates several millilitres of a high titre (~108 pfu/mL) phage lysate.
Protocol
Materials needed:
- Plates containing the bacteriophage grown on a permissive host strain (see Streptomyces bacteriophage plaque assay)
- Liquid DNB medium
- 0.45µm syringe filter
Instructions:
- Take plates from the Streptomyces Bacteriophage plaque assay
- Take the dilution(s) that show almost confluent lysis (You should still be able to distinguish the shapes of individual plaques, but the plate should display as much lysis as possible - to harvest the maximum number of phage. Often it pays to look at the edges of the plates where it looks like there is no growth of the indicator strain – these often display confluent lysis).
- Flood the plates exhibiting almost confluent lysis with 5 ml of Difco Nutrient broth (DNB), and leave at room temperature for 2 hrs.
- Pour the broth off the plate into a Bijoux bottle (or other suitable container).
- Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 108 pfu/mL.
- This phage preparation (phage lysate) should be stored at 4°C.
Notes
- The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
- It is not necessary to add chloroform to the phage lysates.
Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.
