Cesium Chloride Purification of T7 - Revision history

Torsten Waldminghaus at 17:22, 23 February 2009

2009-02-23T17:22:07Z

←Older revision		Revision as of 17:22, 23 February 2009
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		+	{{back to protocols}}
	==Summary==		==Summary==
	Cleaner stocks of T7 that concentrates and purifies T7 bacteriophage.		Cleaner stocks of T7 that concentrates and purifies T7 bacteriophage.

Reshma P. Shetty at 20:16, 8 May 2007

2007-05-08T20:16:12Z

←Older revision		Revision as of 20:16, 8 May 2007
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	Characterization of Bacteriophage T7 DNA Entry into ''Escherichia coli''		Characterization of Bacteriophage T7 DNA Entry into ''Escherichia coli''
		+
		+	[[Category:Protocol]] [[Category:T7]]

Skosuri: /* Summary */

2005-06-03T19:53:13Z

Summary

←Older revision		Revision as of 19:53, 3 June 2005
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	==Summary==		==Summary==
		+	Cleaner stocks of T7 that concentrates and purifies T7 bacteriophage.
		+
	==Protocol==		==Protocol==
	#Grow 100mL of permissive cells to a density of 10<sup>8</sup> to 10<sup>9</sup> cells/ml at 30°C in a rotary shaking water bath. Inoculate the cells with a drop from a master phage stock. Continue to shake cells in the water bath at 30°C until culture clarifies. ''Be careful not to let culture sit for long (>15 minutes) after culture clarifies''.		#Grow 100mL of permissive cells to a density of 10<sup>8</sup> to 10<sup>9</sup> cells/ml at 30°C in a rotary shaking water bath. Inoculate the cells with a drop from a master phage stock. Continue to shake cells in the water bath at 30°C until culture clarifies. ''Be careful not to let culture sit for long (>15 minutes) after culture clarifies''.

Skosuri: /* Protocol */

2005-06-03T19:52:21Z

Protocol

←Older revision		Revision as of 19:52, 3 June 2005
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	#Add NaCl to the lysate to make the final concentration 1 molar. Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant. Gently stir the mixture until the PEG has totally dissolved. Keep lysate on ice for 1 hour.		#Add NaCl to the lysate to make the final concentration 1 molar. Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant. Gently stir the mixture until the PEG has totally dissolved. Keep lysate on ice for 1 hour.
	#* Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris. See [[Studier Lysate Prep]]		#* Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris. See [[Studier Lysate Prep]]
-	#Pellet the phage at 5,000 rpm for 15 min. Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of [[T7 ~~buffer~~]]. Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.	+	#Pellet the phage at 5,000 rpm for 15 min. Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of [[T7 Buffer]]. Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.
	#Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor. Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer. Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.		#Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor. Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer. Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.
	#Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.		#Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.

Skosuri: /* Protocol */

2005-06-03T19:52:08Z

Protocol

←Older revision		Revision as of 19:52, 3 June 2005
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	#Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.		#Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.
	#Remove the phage band from the side of the tube with a syringe.		#Remove the phage band from the side of the tube with a syringe.
-	#Remove the cesium chloride by dialysis against 0.5 to 1 Liter of [[T7 ~~buffer~~]] at 4°C.	+	#Remove the cesium chloride by dialysis against 0.5 to 1 Liter of [[T7 Buffer]] at 4°C.
		+
	==Reference==		==Reference==
	Luís René García		Luís René García

Skosuri at 19:51, 3 June 2005

2005-06-03T19:51:15Z

New page

==Summary==
==Protocol==
#Grow 100mL of permissive cells to a density of 10<sup>8</sup> to 10<sup>9</sup> cells/ml at 30°C in a rotary shaking water bath. Inoculate the cells with a drop from a master phage stock. Continue to shake cells in the water bath at 30°C until culture clarifies. ''Be careful not to let culture sit for long (>15 minutes) after culture clarifies''.
#Add NaCl to the lysate to make the final concentration 1 molar. Centrifuge the lysate at 10,000 rpm for 10 min, Discard the cellular debris, and add 10 grams polyethylene glycol (PEG) m.w. 8000 (10% w/v) to the supernatant. Gently stir the mixture until the PEG has totally dissolved. Keep lysate on ice for 1 hour.
#* Need to check the 1 hour incubation step because of warning from Studier on leaving cultures too long in the cell debris. See [[Studier Lysate Prep]]
#Pellet the phage at 5,000 rpm for 15 min. Decant the supernatant, and very gently resuspend the pellet in 3.5 mL of [[T7 buffer]]. Centrifuge the lysate at 5,000 rpm for 10 min, and keep the supernatant.
#Pour a cesium chloride step gradient: add 0.5 mL of cesium chloride with a density of 1.6 to the bottom of a centrifuge tube that fits in a SW 40.1 rotor. Gently layer 0.5 mL of cesium chloride ρ=1.5 onto the ρ=1.6 layer. Finally add 0.5 mL of cesium chloride ρ=1.4 onto the ρ=1.5 layer.
#Gently layer the phage supernatant onto the cesium chloride step gradient. Centrifuge the phage in a SW 50.1 rotor at 30,000 rpm for 2 to 3 hours. The phage will band at the ρ=1.5 layer.
#Remove the phage band from the side of the tube with a syringe.
#Remove the cesium chloride by dialysis against 0.5 to 1 Liter of [[T7 buffer]] at 4°C.
==Reference==
Luís René García

Ph.D. thesis with Ian Molineux

UT Austin

Characterization of Bacteriophage T7 DNA Entry into ''Escherichia coli''