High titre preparation of phage - Revision history

Paulhoskisson at 12:12, 5 May 2020

2020-05-05T12:12:52Z

← Older revision		Revision as of 12:12, 5 May 2020
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	This protocol has been confirmed to work for the following organisms:		This protocol has been confirmed to work for the following organisms:
	*<em>[[Streptomyces coelicolor]]</em> with ΦC31		*<em>[[Streptomyces coelicolor]]</em> with ΦC31, ΦBT1, ΦHau3, ΦR4
			*<em>[[Streptomyces lividans]]</em> with ΦC31, ΦBT1, ΦHau3, ΦR4
	*<em>[[Streptomyces venezuelae]]</em> with ΦSV1		*<em>[[Streptomyces venezuelae]]</em> with ΦSV1

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	#Take plates from the [[Streptomyces Bacteriophage plaque assay]]		#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. Plates can be gently rocked to increase the phage titre.
	#Pour the broth off the plate into a Bijoux bottle (or other suitable container).		#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 10<sup>8</sup> pfu/mL.		#Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 10<sup>8</sup> pfu/mL.

MFeeney at 10:35, 5 May 2020

2020-05-05T10:35:20Z

← Older revision		Revision as of 10:35, 5 May 2020
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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 ~~bacteriophage~~ plaque assay]]) generates several millilitres of a high titre (~10<sup>8</sup> pfu/mL) phage lysate.		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 ~~bacteriophage~~ plaque assay]]		#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.

MFeeney at 10:23, 5 May 2020

2020-05-05T10:23:58Z

← Older revision		Revision as of 10:23, 5 May 2020
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	#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.
	#Pour off the ~~broth~~ into a Bijoux bottle (or other suitable container).		#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 10<sup>8</sup> pfu/mL.		#Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 10<sup>8</sup> pfu/mL.
	#This phage preparation (phage lysate) should be stored at 4°C.		#This phage preparation (phage lysate) should be stored at 4°C.

MFeeney at 10:21, 5 May 2020

2020-05-05T10:21:50Z

← Older revision		Revision as of 10:21, 5 May 2020
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	Instructions:		Instructions:
	#Take plates from the [[Streptomyces bacteriophage plaque assay]]		#Take plates from the [[Streptomyces bacteriophage plaque assay]]
	#Take the dilution(s) that show almost confluent lysis (~~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.
	#Pour off the broth into a Bijoux bottle (or other suitable container).		#Pour off the broth into a Bijoux bottle (or other suitable container).

MFeeney at 10:19, 5 May 2020

2020-05-05T10:19:13Z

← Older revision		Revision as of 10:19, 5 May 2020
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	===High titre preparation of phage===		===High titre preparation of phage===

	~~It is also useful for the~~ preparation of phage ~~lysates~~ (~~see the protocol, [[High titre preparation of~~ phage]]) ~~which~~ can be used e.g. for generalized transduction or 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).

	~~It can also be used to isolate phage from~~ single ~~plaques~~ (~~the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent~~ a ~~single infection event. (See the protocol,~~ [[~~Isolation of phages from single plaques~~]].)		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===

	Materials needed:		Materials needed:
	# Plates ~~from~~ a [[Streptomyces bacteriophage plaque assay]]		# Plates containing the bacteriophage grown on a permissive host strain (see [[Streptomyces bacteriophage plaque assay]])
	# Liquid [[DNB]] medium		# Liquid [[DNB]] medium
	#0.45µm syringe filter		#0.45µm syringe filter
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	#Take the dilution(s) that show almost confluent lysis (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 (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.
	#Pour off the broth ~~in to~~ a Bijoux bottle ~~and filter~~ the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 10<sup>8</sup> pfu/mL.		#Pour off the broth into a Bijoux bottle (or other suitable container).
	#This phage preparation (phage lysate) ~~can~~ be stored at 4°C.		#Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 10<sup>8</sup> pfu/mL.
			#This phage preparation (phage lysate) should be stored at 4°C.

MFeeney: Created page with "=High titre preparation of ''Streptomyces'' phage= This protocol has been confirmed to work for the following organisms: Streptomyces coelicolor
2020-05-05T10:09:26Z

Created page with "=High titre preparation of ''Streptomyces'' phage= This protocol has been confirmed to work for the following organisms: Streptomyces coelicolor</e..."

New page
=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===

It is also useful for the preparation of phage lysates (see the protocol, [[High titre preparation of phage]]) which can be used e.g. for generalized transduction or other experiments.

It can also be used to isolate phage from single plaques (the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event. (See the protocol, [[Isolation of phages from single plaques]].)

===Protocol===

Materials needed:
# Plates from a [[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 (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 off the broth in to a Bijoux bottle and 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) can 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.