Bacterial cell culture - Revision history

Torsten Waldminghaus at 16:58, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 16:58:14 GMT

←Older revision		Revision as of 16:58, 23 February 2009
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		+	{{back to protocols}}
		+
	==Materials==		==Materials==

Felix Moser: /* Equipment */

Felix Moser — Sun, 06 Jul 2008 23:05:50 GMT

Equipment

←Older revision		Revision as of 23:05, 6 July 2008
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	==Equipment==		==Equipment==
	*Vortexer		*Vortexer
-	*Fireboy or Bunsen burner	+	*[[Fireboy]] or Bunsen burner
	*Motorized pipette		*Motorized pipette
	*Micropipettes and sterile tips		*Micropipettes and sterile tips

John Wertz: /* Procedure */ Removed suggestion to eject pipette tip into culture. This is poor sterile technique.

John Wertz — Tue, 06 May 2008 20:04:40 GMT

Procedure: Removed suggestion to eject pipette tip into culture. This is poor sterile technique.

←Older revision		Revision as of 20:04, 6 May 2008
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	#Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.		#Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.
	#Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.		#Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.
-	#Pick up one colony by tapping a small (0.1 μl) pipette tip (held on a pipette) on the surface of the plate. Uncap a tube, flame the top, ~~eject~~ the tip ~~into~~ the tube, flame, cap. You can also use a sterile toothpick for transfer~~, but this method gives a clear indication of which tubes have been inoculated~~.	+	#Pick up one colony by tapping a small (0.1 μl) pipette tip (held on a pipette) on the surface of the plate. Uncap a tube, flame the top, tip the tube so as to transfer cells from the pipette tip to the surface of the media without touching the inside of the tube with the non-sterile portion of the pipetter, flame, cap. You can also use a sterile toothpick for transfer.
	#*Often people use just a sterile metal loop (sterile by flaming) to place the colony in the tube. This is because flaming assures the sterility of the loop, whereas disposables such as pipette tips and toothpicks can be contaminated, and cannot be flamed on the spot.		#*Often people use just a sterile metal loop (sterile by flaming) to place the colony in the tube. This is because flaming assures the sterility of the loop, whereas disposables such as pipette tips and toothpicks can be contaminated, and cannot be flamed on the spot.
	#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.		#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.

Reshma P. Shetty at 20:18, 8 May 2007

Reshma P. Shetty — Tue, 08 May 2007 20:18:50 GMT

←Older revision		Revision as of 20:18, 8 May 2007
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	[[Category:Protocol]]		[[Category:Protocol]]
		+	[[Category:In vivo]]
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]

Reshma P. Shetty at 22:43, 5 December 2006

Reshma P. Shetty — Tue, 05 Dec 2006 22:43:34 GMT

←Older revision		Revision as of 22:43, 5 December 2006
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	#*The amount of time you wait depends on the reason for growing the cells. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, take care to take readings at the same cell culture density each time.		#*The amount of time you wait depends on the reason for growing the cells. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, take care to take readings at the same cell culture density each time.

-	[[Category:~~E.Coli~~ Protocol]]	+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]

ClarkeS: /* Procedure */ clarity and made more universal, though still very MIT-centered

ClarkeS — Thu, 13 Jul 2006 18:02:42 GMT

Procedure: clarity and made more universal, though still very MIT-centered

←Older revision		Revision as of 18:02, 13 July 2006
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	#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.		#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.
	#Vortex each tube for 1-2 seconds to mix well.		#Vortex each tube for 1-2 seconds to mix well.
-	#Take the tubes to incubate. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the ~~motion is~~ too abrupt. Add your tubes in a balanced layout. If you have an odd number, ~~there are~~ extra tubes for balance ~~in the box on the floor next to the machine~~. Turn the ~~rotating~~ back on to 7. '''Do not forget to turn the rack back on.'''	+	#Take the tubes to incubate. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the stop will be too abrupt. Add your tubes in a balanced layout. If you have an odd number, use extra empty tubes for balance. Turn the rotation back on to 7 (applies to MIT building 68 5th floor warm room). '''Do not forget to turn the rack back on.'''
-	#*Incubation once again is often at 37°C in ~~the temperature controlled~~ room.	+	#*Incubation once again is often at 37°C in an incubator or warm room.
	#Wait overnight or until your cells have reached the desired concentration.		#Wait overnight or until your cells have reached the desired concentration.
-	#*The amount of time ~~that one waits is dependent upon what~~ you ~~are~~ growing the cells ~~to do~~. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, ~~one should~~ take care to take readings at the same cell culture density each time.	+	#*The amount of time you wait depends on the reason for growing the cells. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, take care to take readings at the same cell culture density each time.

	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

ClarkeS: /* Procedure */

ClarkeS — Wed, 22 Mar 2006 20:56:48 GMT

Procedure

←Older revision		Revision as of 20:56, 22 March 2006
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	For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.		For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.

-	#Streak an [[Endy:Pouring plates\|agar plate]] from ~~glyerol~~ stock. Incubate plates until colonies grow.	+	#Streak an [[Endy:Pouring plates\|agar plate]] from glycerol stock. Incubate plates until colonies grow.
	#*Most incubations with ''E. coli'' take place at 37°C. Often bacteria with temperature sensitive mutations need to be grown at 30° instead.		#*Most incubations with ''E. coli'' take place at 37°C. Often bacteria with temperature sensitive mutations need to be grown at 30° instead.
	#*The reason you need to streak plates is to be certain that you are starting from a single clonal population of cells. In this case, colonies that are picked are assumed to arise from a single cell dividing to form the colony.		#*The reason you need to streak plates is to be certain that you are starting from a single clonal population of cells. In this case, colonies that are picked are assumed to arise from a single cell dividing to form the colony.

Barry Canton: /* Procedure */

Barry Canton — Mon, 20 Mar 2006 21:50:26 GMT

Procedure

←Older revision		Revision as of 21:50, 20 March 2006
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	For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.		For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.

-	#Streak an [[~~Pour~~ plates\|agar plate]] from glyerol stock. Incubate plates until colonies grow.	+	#Streak an [[Endy:Pouring plates\|agar plate]] from glyerol stock. Incubate plates until colonies grow.
	#*Most incubations with ''E. coli'' take place at 37°C. Often bacteria with temperature sensitive mutations need to be grown at 30° instead.		#*Most incubations with ''E. coli'' take place at 37°C. Often bacteria with temperature sensitive mutations need to be grown at 30° instead.
	#*The reason you need to streak plates is to be certain that you are starting from a single clonal population of cells. In this case, colonies that are picked are assumed to arise from a single cell dividing to form the colony.		#*The reason you need to streak plates is to be certain that you are starting from a single clonal population of cells. In this case, colonies that are picked are assumed to arise from a single cell dividing to form the colony.

Skosuri: category addition

Skosuri — Fri, 29 Jul 2005 23:47:56 GMT

category addition

←Older revision		Revision as of 23:47, 29 July 2005
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	#Wait overnight or until your cells have reached the desired concentration.		#Wait overnight or until your cells have reached the desired concentration.
	#*The amount of time that one waits is dependent upon what you are growing the cells to do. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, one should take care to take readings at the same cell culture density each time.		#*The amount of time that one waits is dependent upon what you are growing the cells to do. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, one should take care to take readings at the same cell culture density each time.
		+
		+	[[Category:E.Coli Protocol]]

Skosuri at 23:46, 29 July 2005

Skosuri — Fri, 29 Jul 2005 23:46:37 GMT

←Older revision		Revision as of 23:46, 29 July 2005
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	#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.		#Pipette the desired amount of [[Endy:Preparing Antibiotic Stocks\|antibiotic]] into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube.
	#Vortex each tube for 1-2 seconds to mix well.		#Vortex each tube for 1-2 seconds to mix well.
-	#Take the tubes to incubate. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the motion is too abrupt. Add your tubes in a balanced	+	#Take the tubes to incubate. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the motion is too abrupt. Add your tubes in a balanced layout. If you have an odd number, there are extra tubes for balance in the box on the floor next to the machine. Turn the rotating back on to 7. '''Do not forget to turn the rack back on.'''
-	layout. If you have an odd number, there are extra tubes for balance in the box on the floor next to the machine. Turn the rotating back on to 7. '''Do not forget to turn the rack back on.'''	+
	#*Incubation once again is often at 37°C in the temperature controlled room.		#*Incubation once again is often at 37°C in the temperature controlled room.
	#Wait overnight or until your cells have reached the desired concentration.		#Wait overnight or until your cells have reached the desired concentration.
		+	#*The amount of time that one waits is dependent upon what you are growing the cells to do. To [[Miniprep\|miniprep]] plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, one should take care to take readings at the same cell culture density each time.