Lipofectamine Mediated Knockdown - Revision history

Davebrid: fixed typo pmoles should be nmoles

2009-12-18T16:39:45Z

fixed typo pmoles should be nmoles

← Older revision		Revision as of 16:39, 18 December 2009
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	==Required Amounts==		==Required Amounts==
	*Calculate RNA to transfect (typically 40 pmol per well)		*Calculate RNA to transfect (typically 40 pmol per well)
	Lipofectamine(in uL) = RNA(in ~~pmoles~~) 50		Lipofectamine(in uL) = RNA(in nmoles) 50
	OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) 50		OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) 50

Davebrid: rewrote according to lipofectamine instructions

2009-12-18T16:38:45Z

rewrote according to lipofectamine instructions

← Older revision		Revision as of 16:38, 18 December 2009
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	~~__NOTOC__~~		[[Category: Cell Culture ]]
	==~~siRNA Double Transfection of RAW 264.7~~ Cells ~~with Lipofectamine 2000~~ in a ~~6-Well Dish==~~		[[Category: Knockdown]]
	~~AfCS Procedure Protocol PP00000262~~
	~~Version 1, 10/~~12~~/04~~		==Materials==
	The AfCS is utilizing RNA interference (RNAi) to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. One approach to this technique is transient transfection of gene-specific small interfering RNAs (siRNAs). The following procedure involves the transfection of siRNA into RAW 264.7 cells using Lipofectamine 2000~~. Cells are transfected with siRNA twice~~ (~~on subsequent days). Target gene knockdown is assessed from total RNA isolated 48 hr post~~-~~transfection or from protein isolated 72 hr post-transfection~~.		*Cells in a 12 well dish
	==~~=Day 1=~~==		*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
	~~#Count RAW 264.7 cells and plate in a 6-~~well ~~plate at 5 x 105 cells/well, 2.5 ml/well.~~
	~~#Incubate cells at 37 °C~~ in ~~a humidified, 5% CO2 incubator for 24 hr.~~		==Required Amounts==
	~~===Day 2==~~=		*Calculate RNA to transfect (typically 40 pmol per well)
	~~#In an RNase-free tube~~, ~~dilute 10 μl of 40 μM siRNA duplex in a total volume of 250 μl Opti-MEM. Mix gently.~~		Lipofectamine(in uL) = RNA(in pmoles) 50
	~~#Mix~~ Lipofectamine ~~2000 gently before use.~~		OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) 50
	~~#In a separate RNase-free tube, dilute 10 μl~~ Lipofectamine ~~2000~~ in ~~a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.~~
	~~#After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000~~ (~~total volume is 500 μl~~)~~. Mix gently and incubate for 20 min at room temperature.~~		==Protocol (6 well dish)==
	#~~During~~ the ~~20 min incubation, aspirate medium~~ from ~~cells. Add 500 μl fresh RAW 264.7 growth medium 1 (RAWGM1) to cells.~~		#Plate cells the day before from a confluent dish diluted to 1:12 into DMEM/FBS with no PSG
	~~#Add Lipofectamine 2000 complexes~~ to ~~wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of~~ 1 ml/~~well. Mix gently by rocking the plate, and incubate cells for 4 hour at 37 °C in a humidified, 5% CO2 incubator.~~		#For each well prepare, scalin up as required:
	#~~After 4 hr, add 1.5 ml RAWGM1 to~~ each well.		##100uL OptiMEM plus 40 pmoles of RNA.
	~~8. Continue to incubate cells at 37 °C in a humidified~~, ~~5% CO2 incubator.~~		##100uL OptiMEM plus 2 uL of Lipofectamine.
	~~===Day 3===~~		##Incubate ~5 minutes at room temperature.
	#~~In an RNase-free tube, dilute 10 μl of~~ 40 ~~μM duplex in a total volume~~ of ~~250 μl Opti-MEM. Mix gently~~.		##Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine (should be 200uL/well).
	#~~Mix~~ Lipofectamine ~~2000 gently before use~~.		##Incubate ~20 min at room temperature.
	#~~In a separate RNase-free tube, dilute 10 μl Lipofectamine 2000 in a total volume of 250 μl Opti-MEM. Mix gently and incubate for~~ 5 ~~min~~ at room temperature.		#Add DNA/Lipofectamine complexes (~200 uL) to the 1 mL of media on the cells
	#~~After~~ the ~~5 min incubation, combine the diluted duplex with the diluted~~ Lipofectamine ~~2000~~ (~~total volume is 500 μl~~). ~~Mix gently and incubate for~~ 20 min at room temperature.		#Gently rock plate to mix
	~~#During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAWGM1 to cells~~.		#After ~4h re-feed cells with normal media.
	#Add Lipofectamine ~~2000~~ complexes ~~to wells~~ (~~500 μl/well~~)~~. The final siRNA concentration is 400 nM in a final volume~~ of ~~1 ml/well. Mix gently by rocking~~ the ~~plate, and incubate~~ cells ~~for 4 hr at 37 °C in a humidified, 5% CO2 incubator.~~
	#~~After 4 hr, add 1.5 ml RAWGM1~~ to ~~each well.~~		Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual]
	#~~Continue to incubate~~ cells ~~at 37 °C in a humidified, 5% CO2 incubator~~.
	~~===Day 4===~~
	~~#Harvest cells for RNA analysis as indicated in Cell Lysis and Total RNA Isolation~~ from ~~RAW 264.7 Cells, AfCS protocol PP00000260, at approximately 48 hr from start of first transfection.~~
	~~#For cells destined for protein analysis, change medium on cells.~~
	~~#Aspirate medium and add 2.5 ml fresh medium per well.~~
	~~===Day 5===~~
	~~#Harvest cells for protein analysis as indicated in Cell Lysis and Protein Extraction from RAW 264.7 Cells, AfCS protocol PP00000258, at approximately 72 hr from start of first transfection.~~
	~~==Reagents and Materials==~~
	*Cell culture cluster, 6 well: ~~Corning Inc~~.~~; catalog no~~. ~~3516~~
	*Microtube, 1.5 ml, RNase-, DNase-, pyrogen-free: VWR; catalog no. KT749510-1590
	*Opti-MEM I reduced-serum medium (1X) liquid: Invitrogen; catalog no. ~~31985088~~
	*Lipofectamine 2000: Invitrogen; catalog no. 11668019
	*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510
	~~[[ Category:Cell Culture ]]~~
	~~[[ Category:Transfection]~~]

Davebrid at 17:48, 12 August 2009

2009-08-12T17:48:47Z

← Older revision		Revision as of 17:48, 12 August 2009
Line 33:		Line 33:
	==Reagents and Materials==		==Reagents and Materials==
	*Cell culture cluster, 6 well: Corning Inc.; catalog no. 3516		*Cell culture cluster, 6 well: Corning Inc.; catalog no. 3516
	*Microtube, 1.5 ml, RNase-, DNase-, pyrogen-free: VWR; catalog no.		*Microtube, 1.5 ml, RNase-, DNase-, pyrogen-free: VWR; catalog no. KT749510-1590
	KT749510-1590
	*Opti-MEM I reduced-serum medium (1X) liquid: Invitrogen; catalog no. 31985088		*Opti-MEM I reduced-serum medium (1X) liquid: Invitrogen; catalog no. 31985088
	*Lipofectamine 2000: Invitrogen; catalog no. 11668019		*Lipofectamine 2000: Invitrogen; catalog no. 11668019

Davebrid at 17:48, 12 August 2009

2009-08-12T17:48:33Z

← Older revision		Revision as of 17:48, 12 August 2009
Line 1:		Line 1:
			__NOTOC__
	==siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine 2000 in a 6-Well Dish==		==siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine 2000 in a 6-Well Dish==
	AfCS Procedure Protocol PP00000262		AfCS Procedure Protocol PP00000262

Davebrid at 17:48, 12 August 2009

2009-08-12T17:48:21Z

← Older revision		Revision as of 17:48, 12 August 2009
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	*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510		*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510
	[[ Category:Cell Culture ]]		[[ Category:Cell Culture ]]
			[[ Category:Transfection]]

Davebrid at 15:28, 15 July 2009

2009-07-15T15:28:45Z

← Older revision		Revision as of 15:28, 15 July 2009
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	*Lipofectamine 2000: Invitrogen; catalog no. 11668019		*Lipofectamine 2000: Invitrogen; catalog no. 11668019
	*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510		*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510
			[[ Category:Cell Culture ]]

Davebrid: copied initial page

2009-07-01T18:05:06Z

copied initial page

New page

==siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine 2000 in a 6-Well Dish==
AfCS Procedure Protocol PP00000262
Version 1, 10/12/04
The AfCS is utilizing RNA interference (RNAi) to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. One approach to this technique is transient transfection of gene-specific small interfering RNAs (siRNAs). The following procedure involves the transfection of siRNA into RAW 264.7 cells using Lipofectamine 2000. Cells are transfected with siRNA twice (on subsequent days). Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection.
===Day 1===
#Count RAW 264.7 cells and plate in a 6-well plate at 5 x 105 cells/well, 2.5 ml/well.
#Incubate cells at 37 °C in a humidified, 5% CO2 incubator for 24 hr.
===Day 2===
#In an RNase-free tube, dilute 10 μl of 40 μM siRNA duplex in a total volume of 250 μl Opti-MEM. Mix gently.
#Mix Lipofectamine 2000 gently before use.
#In a separate RNase-free tube, dilute 10 μl Lipofectamine 2000 in a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.
#After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 min at room temperature.
#During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAW 264.7 growth medium 1 (RAWGM1) to cells.
#Add Lipofectamine 2000 complexes to wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of 1 ml/well. Mix gently by rocking the plate, and incubate cells for 4 hour at 37 °C in a humidified, 5% CO2 incubator.
#After 4 hr, add 1.5 ml RAWGM1 to each well.
8. Continue to incubate cells at 37 °C in a humidified, 5% CO2 incubator.
===Day 3===
#In an RNase-free tube, dilute 10 μl of 40 μM duplex in a total volume of 250 μl Opti-MEM. Mix gently.
#Mix Lipofectamine 2000 gently before use.
#In a separate RNase-free tube, dilute 10 μl Lipofectamine 2000 in a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.
#After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 min at room temperature.
#During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAWGM1 to cells.
#Add Lipofectamine 2000 complexes to wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of 1 ml/well. Mix gently by rocking the plate, and incubate cells for 4 hr at 37 °C in a humidified, 5% CO2 incubator.
#After 4 hr, add 1.5 ml RAWGM1 to each well.
#Continue to incubate cells at 37 °C in a humidified, 5% CO2 incubator.
===Day 4===
#Harvest cells for RNA analysis as indicated in Cell Lysis and Total RNA Isolation from RAW 264.7 Cells, AfCS protocol PP00000260, at approximately 48 hr from start of first transfection.
#For cells destined for protein analysis, change medium on cells.
#Aspirate medium and add 2.5 ml fresh medium per well.
===Day 5===
#Harvest cells for protein analysis as indicated in Cell Lysis and Protein Extraction from RAW 264.7 Cells, AfCS protocol PP00000258, at approximately 72 hr from start of first transfection.
==Reagents and Materials==
*Cell culture cluster, 6 well: Corning Inc.; catalog no. 3516
*Microtube, 1.5 ml, RNase-, DNase-, pyrogen-free: VWR; catalog no.
KT749510-1590
*Opti-MEM I reduced-serum medium (1X) liquid: Invitrogen; catalog no. 31985088
*Lipofectamine 2000: Invitrogen; catalog no. 11668019
*RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510