Isolation of phages from environmental samples - Revision history

Paulhoskisson: /* Isolation of phages from environmental samples */

2020-05-05T12:22:26Z

Isolation of phages from environmental samples

← Older revision		Revision as of 12:22, 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 lividans]]</em> strain TK24		*<em>[[Streptomyces lividans]]</em> strain TK24
	*<em>[[Streptomyces coelicolor]]</em> strain J1929 (pgl ~~negative~~ strain)		*<em>[[Streptomyces coelicolor]]</em> strain J1929 (pgl<sup>-</sup> strain)

Paulhoskisson: /* Protocol */

2020-05-05T12:22:03Z

Protocol

← Older revision		Revision as of 12:22, 5 May 2020
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	Enrichment -		Enrichment -
	#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.		#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
	#Add 500 µl of a dense spore preparation (1 x 10<sup>9</sup>) from a Streptomyces indicator strain of your choice - see note 1.		#Add 500 µl of a dense spore preparation (~1 x 10<sup>9</sup>) from a Streptomyces indicator strain of your choice - see note 1.
	#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.		#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
	#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.		#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.

Paulhoskisson: /* Protocol */

2020-05-05T12:21:45Z

Protocol

← Older revision		Revision as of 12:21, 5 May 2020
Line 25:		Line 25:
	Enrichment -		Enrichment -
	#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.		#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
	#Add 500 µl of a dense spore preparation (1 x 10 <sup>9</sup>) from a Streptomyces indicator strain of your choice - see note 1.		#Add 500 µl of a dense spore preparation (1 x 10<sup>9</sup>) from a Streptomyces indicator strain of your choice - see note 1.
	#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.		#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
	#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.		#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.

Paulhoskisson: /* Protocol */

2020-05-05T12:21:25Z

Protocol

← Older revision		Revision as of 12:21, 5 May 2020
Line 25:		Line 25:
	Enrichment -		Enrichment -
	#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.		#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
	#Add 500 µl of a dense spore preparation (1 x 10 <sup>9<sup>) from a Streptomyces indicator strain of your choice - see note 1.		#Add 500 µl of a dense spore preparation (1 x 10 <sup>9</sup>) from a Streptomyces indicator strain of your choice - see note 1.
	#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.		#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
	#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.		#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
	#Decant the enriched DNB and filter through a 0.45 µm syringe filter.		#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
	#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.		#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.



	===Notes===		===Notes===

Paulhoskisson: /* Isolation of phages from environmental samples */

2020-05-05T12:20:33Z

Isolation of phages from environmental samples

← Older revision		Revision as of 12:20, 5 May 2020
Line 25:		Line 25:
	Enrichment -		Enrichment -
	#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.		#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
	#Add 500 µl of a dense spore preparation (1 x 10 <sup>9<sup>)from a Streptomyces indicator strain of your choice - see note 1.		#Add 500 µl of a dense spore preparation (1 x 10 <sup>9<sup>) from a Streptomyces indicator strain of your choice - see note 1.
	#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.		#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
	#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.		#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.

Paulhoskisson at 12:19, 5 May 2020

2020-05-05T12:19:55Z

← Older revision		Revision as of 12:19, 5 May 2020
Line 3:		Line 3:
	This protocol has been confirmed to work for the following organisms:		This protocol has been confirmed to work for the following organisms:
	*<em>[[Streptomyces lividans]]</em> strain TK24		*<em>[[Streptomyces lividans]]</em> strain TK24
			*<em>[[Streptomyces coelicolor]]</em> strain J1929 (pgl negative strain)


	===Isolation of phages from environmental samples===		===Isolation of phages from environmental samples===
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	Enrichment -		Enrichment -
	#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.		#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
	#Add 500 µl of a dense spore preparation from a Streptomyces indicator strain of your choice - see note 1.		#Add 500 µl of a dense spore preparation (1 x 10 <sup>9<sup>)from a Streptomyces indicator strain of your choice - see note 1.
	#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.		#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
	#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.		#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.

MFeeney at 11:11, 5 May 2020

2020-05-05T11:11:17Z

← Older revision		Revision as of 11:11, 5 May 2020
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	It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)		It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

	This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1).		This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

	===Protocol===		===Protocol===

MFeeney: Created page with "=Isolation of phages from environmental samples= This protocol has been confirmed to work for the following organisms: **Streptomyces lividans* s..."

2020-05-05T11:07:24Z

Created page with "=Isolation of phages from environmental samples= This protocol has been confirmed to work for the following organisms: *Streptomyces lividans s..."

New page

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1).

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples - Revision history

Paulhoskisson: /* Isolation of phages from environmental samples */

Paulhoskisson: /* Protocol */

Paulhoskisson: /* Protocol */

Paulhoskisson: /* Protocol */

Paulhoskisson: /* Isolation of phages from environmental samples */

Paulhoskisson at 12:19, 5 May 2020

MFeeney at 11:11, 5 May 2020

MFeeney: Created page with "=Isolation of phages from environmental samples= This protocol has been confirmed to work for the following organisms: *Streptomyces lividans s..."

MFeeney: Created page with "=Isolation of phages from environmental samples= This protocol has been confirmed to work for the following organisms: **Streptomyces lividans* s..."