Haynes:TransfectionPlasmid Lipo - Revision history

Karmella Haynes at 22:22, 14 May 2013

Karmella Haynes — Tue, 14 May 2013 22:22:16 GMT

←Older revision		Revision as of 22:22, 14 May 2013
Line 1:		Line 1:
	<- [[Haynes:Protocols \| Back to Protocols]]		<- [[Haynes:Protocols \| Back to Protocols]]

-	<div style="width: ~~800px~~">	+	<div style="width: 1000px">

	=Mammalian Cell Transfection - Lipofectamine LTX Reagent=		=Mammalian Cell Transfection - Lipofectamine LTX Reagent=

Karmella Haynes: /* Materials */

Karmella Haynes — Tue, 14 May 2013 21:33:23 GMT

Materials

←Older revision		Revision as of 21:33, 14 May 2013
Line 15:		Line 15:


-	==Procedure - 6-well Format==~~<br>~~	+	==Procedure - 6-well Format==

	'''1 day before transfection:'''<br>		'''1 day before transfection:'''<br>

Karmella Haynes at 21:33, 14 May 2013

Karmella Haynes — Tue, 14 May 2013 21:33:11 GMT

←Older revision		Revision as of 21:33, 14 May 2013
Line 1:		Line 1:
	<- [[Haynes:Protocols \| Back to Protocols]]		<- [[Haynes:Protocols \| Back to Protocols]]
		+
		+	<div style="width: 800px">

	=Mammalian Cell Transfection - Lipofectamine LTX Reagent=		=Mammalian Cell Transfection - Lipofectamine LTX Reagent=
-	~~<div style="width: 800px" align="center">~~

	Karmella Haynes, 2012		Karmella Haynes, 2012
-	~~</div>~~

	==Materials==		==Materials==
-	~~<div style="width: 800px">~~

	* Lipofectamine LTX		* Lipofectamine LTX
Line 16:		Line 15:


-	~~'''~~6-~~WELL FORMAT'''~~<br>	+	==Procedure - 6-well Format==<br>

	'''1 day before transfection:'''<br>		'''1 day before transfection:'''<br>

Karmella Haynes at 21:32, 14 May 2013

Karmella Haynes — Tue, 14 May 2013 21:32:11 GMT

←Older revision		Revision as of 21:32, 14 May 2013
Line 1:		Line 1:
	<- [[Haynes:Protocols \| Back to Protocols]]		<- [[Haynes:Protocols \| Back to Protocols]]

		+	=Mammalian Cell Transfection - Lipofectamine LTX Reagent=
	<div style="width: 800px" align="center">		<div style="width: 800px" align="center">
-	~~'''Mammalian Cell Transfection - Lipofectamine LTX Reagent'''<br>~~	+
	Karmella Haynes, 2012		Karmella Haynes, 2012
	</div>		</div>

		+	==Materials==
	<div style="width: 800px">		<div style="width: 800px">

-	~~'''Materials'''~~
	* Lipofectamine LTX		* Lipofectamine LTX
	* Opti-MEM I Reduced serum medium		* Opti-MEM I Reduced serum medium

Karmella Haynes at 19:54, 7 January 2013

Karmella Haynes — Mon, 07 Jan 2013 19:54:26 GMT

←Older revision		Revision as of 19:54, 7 January 2013
Line 13:		Line 13:
	* Antibiotic-free growth medium (no pen/strep or other antibiotics)		* Antibiotic-free growth medium (no pen/strep or other antibiotics)
	* Cells		* Cells
		+

	'''6-WELL FORMAT'''<br>		'''6-WELL FORMAT'''<br>

Karmella Haynes at 19:54, 7 January 2013

Karmella Haynes — Mon, 07 Jan 2013 19:54:12 GMT

←Older revision		Revision as of 19:54, 7 January 2013
Line 14:		Line 14:
	* Cells		* Cells

-	'''6-~~well format~~'''<br>	+	'''6-WELL FORMAT'''<br>

	'''1 day before transfection:'''<br>		'''1 day before transfection:'''<br>

Karmella Haynes at 23:34, 30 November 2012

Karmella Haynes — Fri, 30 Nov 2012 23:34:30 GMT

←Older revision		Revision as of 23:34, 30 November 2012
Line 24:		Line 24:
	## Label sterile microfuge (1.5 ml) tubes.		## Label sterile microfuge (1.5 ml) tubes.
	## For each transfection, bring '''2.0 μg plasmid DNA''' up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.		## For each transfection, bring '''2.0 μg plasmid DNA''' up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
-	## Add '''~~580~~ μL Opti-MEM''' to each 20 μL DNA sample.	+	## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
	## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.		## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
	## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.		## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.

Karmella Haynes at 03:01, 17 November 2012

Karmella Haynes — Sat, 17 Nov 2012 03:01:22 GMT

←Older revision		Revision as of 03:01, 17 November 2012
Line 29:		Line 29:
	# Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells.		# Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells.
	# Incubate cells at 37°C in a CO<sub>2</sub> incubator		# Incubate cells at 37°C in a CO<sub>2</sub> incubator
-	# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess ~~Lipo-~~DNA complexes. Add fresh antibiotic-free growth medium.	+	# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

	Transgene expression should be detectable after 24 hours.		Transgene expression should be detectable after 24 hours.

Karmella Haynes at 03:00, 17 November 2012

Karmella Haynes — Sat, 17 Nov 2012 03:00:59 GMT

←Older revision		Revision as of 03:00, 17 November 2012
Line 2:		Line 2:

	<div style="width: 800px" align="center">		<div style="width: 800px" align="center">
-	'''Mammalian Cell Transfection - Lipofectamine Reagent'''<br>	+	'''Mammalian Cell Transfection - Lipofectamine LTX Reagent'''<br>
	Karmella Haynes, 2012		Karmella Haynes, 2012
	</div>		</div>

	<div style="width: 800px">		<div style="width: 800px">
-	~~Note: Good for quick-and-dirty transfections; pretty toxic~~

	'''Materials'''		'''Materials'''
-	* Lipofectamine ~~2000~~	+	* Lipofectamine LTX
	* Opti-MEM I Reduced serum medium		* Opti-MEM I Reduced serum medium
	* Antibiotic-free growth medium (no pen/strep or other antibiotics)		* Antibiotic-free growth medium (no pen/strep or other antibiotics)
		+	* Cells

	'''6-well format'''<br>		'''6-well format'''<br>

	'''1 day before transfection:'''<br>		'''1 day before transfection:'''<br>
-	# Seed cultures in 6-well plates at ~2.~~5x105~~ so that cells will be ~5.~~0x105~~ at the time of transfection. Use antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep).	+	# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well.

	'''Transfection:'''<br>		'''Transfection:'''<br>
-	# Warm plasmid DNA, Lipofectamine ~~2000~~, and Opti-MEM I Reduced Serum Medium to room ~~temp~~.	+	# Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
-	# Prepare DNA-Lipofectamine complexes (~~500~~ μl per ~~well~~) as follows:	+	# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
	## Label sterile microfuge (1.5 ml) tubes.		## Label sterile microfuge (1.5 ml) tubes.
-	## ~~Dilute~~ '''plasmid DNA ~~(4 μg)~~''' ~~in 250 μl of~~ Opti-MEM in each ~~tube~~.	+	## For each transfection, bring '''2.0 μg plasmid DNA''' up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
-	## ~~Dilute 4~~.~~0 μl Lipofectamine 2000 in 250 μl of~~ Opti-MEM ~~(per~~ sample). Incubate for 5 minutes at room ~~temp. Do not let the mixture sit for more than 25 minutes~~.	+	## Add '''580 μL Opti-MEM''' to each 20 μL DNA sample.
-	## Add ~~50 μl of the diluted~~ Lipofectamine ~~2000~~ to ~~the diluted~~ DNA ~~(total volume = 100 μl)~~. ~~Mix gently and incubate~~ for 20 minutes at room ~~temp. Complexes are stable for 6 hours at room temp~~.	+	## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
-	## Add ~~500 μl of complexes~~ to each well~~. Mix with gentle rocking~~.	+	## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
-	# Incubate cells at 37°C in a ~~CO2~~ incubator ~~for~~ 5-6 hours~~. Wash~~ cells once with warm 1xPBS. Add fresh antibiotic-free growth medium. Transgene expression should be detectable after 24 hours.	+	# Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells.
		+	# Incubate cells at 37°C in a CO<sub>2</sub> incubator
		+	# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess Lipo-DNA complexes. Add fresh antibiotic-free growth medium.
		+
		+	Transgene expression should be detectable after 24 hours.

	<br><br>		<br><br>
	''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.		''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.

Karmella Haynes at 02:42, 17 November 2012

Karmella Haynes — Sat, 17 Nov 2012 02:42:45 GMT

←Older revision		Revision as of 02:42, 17 November 2012
Line 22:		Line 22:
	# Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.		# Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.
	# Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:		# Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:
-	## Label sterile microfuge ml tubes.	+	## Label sterile microfuge (1.5 ml) tubes.
-	## Dilute plasmid DNA (4 μg) in 250 μl of Opti-MEM in each tube.	+	## Dilute '''plasmid DNA (4 μg)''' in 250 μl of Opti-MEM in each tube.
	## Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.		## Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.
	## Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.		## Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.