Endy:Pouring plates - Revision history

Reshma P. Shetty at 15:38, 14 March 2007

2007-03-14T15:38:44Z

←Older revision		Revision as of 15:38, 14 March 2007
Line 39:		Line 39:
	#Store the labeled bags of plates in the cold room.		#Store the labeled bags of plates in the cold room.

-	[[Category:~~Miscellaneous~~ Protocol]]	+	[[Category:Protocol]]

Cconboy at 19:37, 19 June 2006

2006-06-19T19:37:16Z

←Older revision		Revision as of 19:37, 19 June 2006
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		+	This is a general protocol is for the standard preparation of plates.
		+
		+	Also see the [[Endy:Pouring LB plates from prepped media\|shortcut method]] for using pre-made 1.2% Agar in LB from the Building 68 media prep room.
		+
	==Notes==		==Notes==

-	*~~Melt LB ahead of time because it cools~~ to 55 ~~degrees slowly~~. ~~A good way~~ to ~~do this is melt it in~~ the ~~morning then leave it in the 60 degree incubator or~~ a ~~60 degree bath. If in a rush, do not attempt to cool the media rapidly by running cold water over the container as this can cause the glass to shatter~~. ~~Instead, media can be left at room temperature to cool more rapidly.~~	+	*(Optional) pre-heat a H2O bath to 55 deg C.
		+	*If you don't know how to use the autoclave, ask for a demo.

	==Materials==		==Materials==
-		+	For 1 L of Media:
-	*LB Agar 1.2% (~~from the media room~~)	+	*10 g of Agar (for 1% Agar plates (w/v))
-	*~~Empty plates (above any~~ of ~~the -20 freezers)~~	+	*media components specific to media of interest
-	*~~antibiotics~~ (~~liquid stocks from our -20 freezer~~)	+	*1 L ddH2O
		+	*Sterile antibiotic stock solution (if required)

	==Method==		==Method==
-	===~~Preparation of LB Agar~~===	+	===Mixing and Sterilizing Media===
-		+	#Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.)
-	#~~Obtain 1~~.2~~% LB agar from the~~ media ~~room (500 ml per bottle~~).	+	#(optional, really unnecessary) add a magnetic stir bar and stir the media.
-	#*Be sure to sign it out	+	#Cover the top of the flask LOOSELY with aluminum foil.
-	~~#Melt in microwave:~~	+	#Autoclave the media on the Fluid Cycle, 30 min, 255 deg C. (Keep in mind autoclave reduces pressure slowly. It will take more time than 30 min to execute the cycle.)
-	~~#*loosen the cap~~	+	#Remove media to 55 deg. H2O bath or room temp to cool.
-	~~#*use 50% power~~ (~~enter time~~, ~~press Power, 5, Start~~)	+	#Once media has cooled to ~55 deg. C, add antibiotics, if required.
-	#*monitor as you melt	+
-	~~#*takes approx. 10 minutes per bottle~~.	+
-	#~~If too hot, let~~ the ~~melted solution cool so that it's warm~~, ~~but not hot~~, to the ~~touch - you can use the water bath~~ to ~~do this (50-60 degrees); this won't let it resolidify~~. At room temp~~. it may take 20 mins for LB~~ to cool ~~sufficiently~~.	+
-	#Once ~~it's cool enough (50-60 degrees) -~~ add antibiotics~~: final concentration~~, ~~50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:~~	+
-	~~#*500μL Amp stock~~	+
-	~~#*1mL Kan stock~~	+
-	~~#Swirl to mix; try not to make many bubbles~~.	+

-	===~~Actual~~ Pouring===	+	===Pouring Plates===

-	#Obtain ~~a container~~ of empty plates. ~~One bottle (500ml)~~ of ~~LB Agar~~ will make about ~~one container of~~ plates ~~(20 plates)~~.	+	#Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
-	#Using sterile technique ~~(flame the top of the bottle)(why? what are you killing here? the plastic sealing ring?)~~, pour the ~~LB Agar~~ into the plates.	+	#Using sterile technique, pour the media into the plates.
	#*Cover the base of the plate, and then just a bit more after that.		#*Cover the base of the plate, and then just a bit more after that.
	#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.		#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.

Barry Canton: Pour plates moved to Endy:Pouring plates

2006-03-20T21:49:11Z

Pour plates moved to Endy:Pouring plates

←Older revision

Revision as of 21:49, 20 March 2006

Hkeller at 22:42, 18 November 2005

2005-11-18T22:42:12Z

←Older revision		Revision as of 22:42, 18 November 2005
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	==Notes==		==Notes==

-	*Melt LB ahead of time because it cools to 55 degrees slowly. A good way to do this is melt it in the morning then leave it in the 60 degree incubator or a 60 degree bath. If in a rush, ~~carefully~~ cool by running cold water over the container.	+	*Melt LB ahead of time because it cools to 55 degrees slowly. A good way to do this is melt it in the morning then leave it in the 60 degree incubator or a 60 degree bath. If in a rush, do not attempt to cool the media rapidly by running cold water over the container as this can cause the glass to shatter. Instead, media can be left at room temperature to cool more rapidly.

	==Materials==		==Materials==

Smoore: /* Actual Pouring */

2005-10-06T15:24:16Z

Actual Pouring

←Older revision		Revision as of 15:24, 6 October 2005
Line 28:		Line 28:

	#Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).		#Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).
-	#Using sterile technique (flame the top of the bottle), pour the LB Agar into the plates.	+	#Using sterile technique (flame the top of the bottle)(why? what are you killing here? the plastic sealing ring?), pour the LB Agar into the plates.
	#*Cover the base of the plate, and then just a bit more after that.		#*Cover the base of the plate, and then just a bit more after that.
-	#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this).	+	#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.
	#Leave plates to dry and cool for a while (overnight even).		#Leave plates to dry and cool for a while (overnight even).
	#*It is a good idea to label the stack of plates to indicate antibiotic.		#*It is a good idea to label the stack of plates to indicate antibiotic.

ClarkeS: /* Actual Pouring */ typos, formatting

2005-08-02T17:29:57Z

Actual Pouring: typos, formatting

ClarkeS at 16:28, 2 August 2005

2005-08-02T16:28:33Z

←Older revision		Revision as of 16:28, 2 August 2005
Line 10:		Line 10:

	==Method==		==Method==
-	===~~Preparatation~~ of LB Agar===	+	===Preparation of LB Agar===

-	#Obtain 1.2% agar from the media room (500 ml per bottle).	+	#Obtain 1.2% LB agar from the media room (500 ml per bottle).
	#*Be sure to sign it out		#*Be sure to sign it out
	#Melt in microwave:		#Melt in microwave:
-	~~#*use 50% power~~
	#*loosen the cap		#*loosen the cap
		+	#*use 50% power (enter time, press Power, 5, Start)
	#*monitor as you melt		#*monitor as you melt
	#*takes approx. 10 minutes per bottle.		#*takes approx. 10 minutes per bottle.
	#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.		#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.
	#Once it's cool enough (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:		#Once it's cool enough (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:
-	#*~~500uL~~ Amp stock	+	#*500μL Amp stock
	#*1mL Kan stock		#*1mL Kan stock
	#Swirl to mix; try not to make many bubbles.		#Swirl to mix; try not to make many bubbles.

Jason R. Kelly at 22:11, 3 May 2005

2005-05-03T22:11:30Z

←Older revision		Revision as of 22:11, 3 May 2005
Line 20:		Line 20:
	#*takes approx. 10 minutes per bottle.		#*takes approx. 10 minutes per bottle.
	#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.		#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.
-	#~~ONCE IT~~'~~S COOL ENOUGH~~ (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:	+	#Once it's cool enough (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar use:
	#*500uL Amp stock		#*500uL Amp stock
	#*1mL Kan stock		#*1mL Kan stock

Jason R. Kelly at 22:11, 3 May 2005

2005-05-03T22:11:04Z

Jason R. Kelly at 22:08, 3 May 2005

2005-05-03T22:08:14Z

←Older revision		Revision as of 22:08, 3 May 2005
Line 20:		Line 20:
	#*takes approx. 10 minutes per bottle.		#*takes approx. 10 minutes per bottle.
	#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.		#If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.
-	#ONCE IT'S COOL ENOUGH (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) ~~(This information posted on the refrigerator with our stuff and antibiotics).~~ For 500mL of LB Agar, use 500uL Amp or 1mL Kan.	+	#ONCE IT'S COOL ENOUGH (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar:
		+	#*use 500uL Amp
		+	#*1mL Kan.
	#Swirl to mix; try not to make many bubbles.		#Swirl to mix; try not to make many bubbles.

←Older revision		Revision as of 17:29, 2 August 2005
Line 27:		Line 27:
	===Actual Pouring===		===Actual Pouring===

-	#Obtain a container of empty plates. 1 bottle (500ml) of LB Agar will make about 1 container of plates (20 plates).	+	#Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).
-	#Using sterile technique, (flame the top of the bottle), pour the LB Agar into the plates.	+	#Using sterile technique (flame the top of the bottle), pour the LB Agar into the plates.
	#*Cover the base of the plate, and then just a bit more after that.		#*Cover the base of the plate, and then just a bit more after that.
-	#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e. more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this).	+	#Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this).
	#Leave plates to dry and cool for a while (overnight even).		#Leave plates to dry and cool for a while (overnight even).
-	#*~~Its~~ a good idea to label the stack of plates to indicate antibiotic.	+	#*It is a good idea to label the stack of plates to indicate antibiotic.
	#Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).		#Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
	#Label the bags following taping rules:		#Label the bags following taping rules:
	#*Red tape in back (indicates LB)		#*Red tape in back (indicates LB)
-	#*Tape in front indicates antibiotic. (~~Green:~~ Kan~~. Yellow:~~ Amp~~.) (More~~ taping/color rules are on the refrigerator in 68-564)	+	#*Tape in front indicates antibiotic (green=Kan, yellow=Amp, more taping/color rules are on the refrigerator in 68-564)
-	#*~~also~~ write name of antibiotic, and concentration, on the front piece of tape.	+	#*Also write name of antibiotic, and concentration, on the front piece of tape.
	#Store the labeled bags of plates in the cold room.		#Store the labeled bags of plates in the cold room.

	[[Category:Miscellaneous Protocol]]		[[Category:Miscellaneous Protocol]]