IGEM:Virginia 2012/Protocols/Phage Isolation - Revision history

Jacqueline K. Grimm at 20:59, 31 May 2012

Jacqueline K. Grimm — Thu, 31 May 2012 20:59:35 GMT

←Older revision		Revision as of 20:59, 31 May 2012
Line 17:		Line 17:
	# 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.
		+	# 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.		# 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.

Alex Zorychta: /* Procedure */

Alex Zorychta — Wed, 30 May 2012 17:41:28 GMT

Procedure

←Older revision		Revision as of 17:41, 30 May 2012
Line 20:		Line 20:
	# 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.
-	# 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.	+	# 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 (10 krpm, 10 min)		# 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.

Alex Zorychta: /* Procedure */

Alex Zorychta — Wed, 30 May 2012 17:36:45 GMT

Procedure

←Older revision		Revision as of 17:36, 30 May 2012
Line 17:		Line 17:
	# 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 [http://openwetware.org/wiki/IGEM:~~Virginia 2012~~/Protocols/PEG/~~NaCl Preparation~~ PEG/NaCl] (probably 20 mL) to the conical 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.
	# 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.

Alex Zorychta: /* Procedure */

Alex Zorychta — Wed, 30 May 2012 17:36:12 GMT

Procedure

←Older revision		Revision as of 17:36, 30 May 2012
Line 17:		Line 17:
	# 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.	+	# 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.
	# 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.

Joseph J. Muldoon at 16:46, 30 May 2012

Joseph J. Muldoon — Wed, 30 May 2012 16:46:49 GMT

←Older revision		Revision as of 16:46, 30 May 2012
Line 20:		Line 20:
	# 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 500μl of SM buffer.	+	# 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.	+	# 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

Jacqueline K. Grimm at 20:50, 29 May 2012

Jacqueline K. Grimm — Tue, 29 May 2012 20:50:51 GMT

←Older revision		Revision as of 20:50, 29 May 2012
Line 7:		Line 7:
	*Chloroform		*Chloroform
	*Conical tube		*Conical tube
-	*PEG/NaCl	+	*20% PEG / 2.5 M NaCl
	*SM buffer		*SM buffer

Line 13:		Line 13:

	# 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 ~~125~~. Pipette this amount of chloroform into the Erlenmeyer flask.	+	# 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.

Jacqueline K. Grimm at 17:18, 29 May 2012

Jacqueline K. Grimm — Tue, 29 May 2012 17:18:53 GMT

←Older revision		Revision as of 17:18, 29 May 2012
Line 24:		Line 24:
	# 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 ~~plaque assay to determine the~~ [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Plaque_Assay phage titer].	+	# Perform a [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Plaque_Assay plaque assay] to determine the phage titer.

	==Notes==		==Notes==

Jacqueline K. Grimm: 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...

Jacqueline K. Grimm — Mon, 28 May 2012 17:11:10 GMT

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...

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 Erlenmeyer flask from the incubator.
# To find the amount of chloroform to add, divide the volume of the culture in the Erlenmeyer flask by 125. 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.
# Resuspend the pellet in 500μl of SM buffer.
# Spin down, and collect the supernatant.
# 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 [http://openwetware.org/wiki/IGEM:Virginia_2012/Protocols/Plaque_Assay 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, [[Talk:{{PAGENAME}}|discuss this protocol]].


[[Category:Protocol]]

IGEM:Virginia 2012/Protocols/Phage Isolation - Revision history

Jacqueline K. Grimm at 20:59, 31 May 2012

Alex Zorychta: /* Procedure */

Alex Zorychta: /* Procedure */

Alex Zorychta: /* Procedure */

Joseph J. Muldoon at 16:46, 30 May 2012

Jacqueline K. Grimm at 20:50, 29 May 2012

Jacqueline K. Grimm at 17:18, 29 May 2012

Jacqueline K. Grimm: 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...

Jacqueline K. Grimm: 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...