Knight:RNA electrophoresis/Denaturing - Revision history

Reshma P. Shetty at 16:50, 6 December 2006

2006-12-06T16:50:44Z

←Older revision		Revision as of 16:50, 6 December 2006
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	==Overview==		==Overview==

-	Electrophoresis permits assessment of RNA by size and amount. In general, electrophoresis of RNA is done as a step prior to Northern analysis. However, this protocol is for visualizing the RNA in the gel.	+	Electrophoresis permits assessment of RNA by size and amount. In general, electrophoresis of RNA is done as a step prior to Northern analysis. However, this protocol is for visualizing the RNA in the gel. Since this is a denaturing protocol, the RNA can be assessed both for quality and size.

	==Materials==		==Materials==

Reshma P. Shetty: Knight:RNA electrophoresis moved to Knight:RNA electrophoresis/Denaturing

2006-12-06T16:47:32Z

Knight:RNA electrophoresis moved to Knight:RNA electrophoresis/Denaturing

←Older revision

Revision as of 16:47, 6 December 2006

Reshma P. Shetty at 23:28, 5 December 2006

2006-12-05T23:28:16Z

←Older revision		Revision as of 23:28, 5 December 2006
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	==Notes==		==Notes==
		+	*[[Avoiding RNase contamination\|Avoid RNase contamination]]

	==References==		==References==

Reshma P. Shetty at 23:05, 5 December 2006

2006-12-05T23:05:41Z

←Older revision		Revision as of 23:05, 5 December 2006
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		+	<font color=red>in progress! has not been tested</font>
		+
	==Overview==		==Overview==

Reshma P. Shetty at 22:53, 5 December 2006

2006-12-05T22:53:11Z

←Older revision		Revision as of 22:53, 5 December 2006
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	#MolecularCloning [[doi:10.1101/pdb.prot4057\|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]]		#MolecularCloning [[doi:10.1101/pdb.prot4057\|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]]
	</biblio>		</biblio>
		+
		+	[[Category:Protocol]]
		+	[[Category:RNA]]
		+	[[Category:In vitro]]

Reshma P. Shetty at 22:18, 5 December 2006

2006-12-05T22:18:46Z

←Older revision		Revision as of 22:18, 5 December 2006
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	*RNA size marker		*RNA size marker
	*RNA gel loading buffer		*RNA gel loading buffer
		+	**95% deionized formamide
		+	***Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
		+	**0.025% (w/v) bromophenol blue
		+	**0.025% (w/v) xylene cyanol FF
		+	**5 mM EDTA (pH 8.0)
		+	**0.025% (w/v) SDS

	*[[SYBR Gold]]		*[[SYBR Gold]]
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	Divide into small aliquots and store at -70°C.		Divide into small aliquots and store at -70°C.
		+
		+	===Prepare RNA gel loading buffer===
		+	#Mix the following
		+	#*95% deionized formamide
		+	#**Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
		+	#*0.025% (w/v) bromophenol blue
		+	#*0.025% (w/v) xylene cyanol FF
		+	#*5 mM EDTA (pH 8.0)
		+	#*0.025% (w/v) SDS

	===Denature RNA samples===		===Denature RNA samples===
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	#Place gel in chamber.		#Place gel in chamber.
	#Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.		#Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.
		+
		+	===Run gel===
		+	#Add 1-2μL RNA gel loading buffer to glyoxylated RNA samples
		+	#Immediately load samples. Leave two outside lanes on each side empty.
		+	#Load RNA size markers on outside lanes of gel.
		+	#Electrophorese at 5V/cm.
		+
		+	===Stain gel===
		+	#Prepare fresh 1:10,000 dilution in RNase free water of SYBR gold.
		+	#Ensure pH is 7.0-8.5.
		+	#Pour into staining tray.
		+	#Place gel in plastic staining container.
		+	#Shield from light.
		+	#Agitate gently for 10-40 mins at room temperature.
		+	#Image with [[Knight:Gel imager]]. (Place a clear ruler next to gel to more accurately assess length.)

	==Notes==		==Notes==

Reshma P. Shetty at 21:31, 5 December 2006

2006-12-05T21:31:31Z

←Older revision		Revision as of 21:31, 5 December 2006
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	==Materials==		==Materials==
	===Reagents===		===Reagents===
		+	*RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
		+	**Add DEPC to final concentration of 0.1%.
		+	**Incubate 1hr at 37°C.
		+	**Autoclave for 15 mins at 15 psi.
	*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)		*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
	**100 mM PIPES		**100 mM PIPES
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	**1.2mL of 10X BPTE electrophoresis buffer		**1.2mL of 10X BPTE electrophoresis buffer
	**0.6mL of 80% glycerol		**0.6mL of 80% glycerol
		+	*RNA size marker
	*RNA gel loading buffer		*RNA gel loading buffer
-	*RNAse free water (make lots for rinsing glassware and electrophoresis chambers)	+
-	**Add DEPC to final concentration of 0.1%.	+
-	**Incubate 1hr at 37°C.	+
-	**Autoclave for 15 mins at 15 psi.	+
	*[[SYBR Gold]]		*[[SYBR Gold]]

	===Equipment===		===Equipment===
		+	*37 °C incubator
		+	*55 °C water bath
		+	*Ice water bath
	*Electrophoresis apparatus		*Electrophoresis apparatus

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	===Prepare BPTE electrophoresis buffer===		===Prepare BPTE electrophoresis buffer===
-	#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O	+	#Prepare 10X buffer by adding the following to 90 ml of distilled H<sub>2</sub>O
	#*3 g of PIPES (free acid)		#*3 g of PIPES (free acid)
	#*6 g of Bis-Tris (free base)		#*6 g of Bis-Tris (free base)
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	#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C		#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
	#Autoclave.		#Autoclave.
		+
		+	Dilute 10X buffer 10-fold with RNase free H<sub>2</sub>O.

	===Prepare glyoxal reaction mixture===		===Prepare glyoxal reaction mixture===
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	Divide into small aliquots and store at -70°C.		Divide into small aliquots and store at -70°C.
		+
		+	===Denature RNA samples===
		+	#Mix 10 μL glyoxal reaction mixture with 1-2 μL RNA (up to 10 μg).
		+	#Also mix 10 μL glyoxal reaction mixture with RNA size marker.
		+	#Incubate RNA samples at 55°C for 1 hr.
		+	#Chill RNA samples for 10 mins in ice water.
		+	#Centrifuge 5 seconds to collect liquid at the bottom of the tubes.
		+
		+	===Cast gel===
		+	Do this step during 1 hr denaturation of samples.
		+
		+	#Cast 1.5% agarose gel in 1X BPTE electrophoresis buffer.
		+	#Use a comb with at least 4 extra lanes for size markers and running dyes.
		+	#Place gel in chamber.
		+	#Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.

	==Notes==		==Notes==

Reshma P. Shetty at 21:23, 5 December 2006

2006-12-05T21:23:34Z

←Older revision		Revision as of 21:23, 5 December 2006
Line 4:		Line 4:

	==Materials==		==Materials==
		+	===Reagents===
	*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)		*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
	**100 mM PIPES		**100 mM PIPES
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	**Autoclave for 15 mins at 15 psi.		**Autoclave for 15 mins at 15 psi.
	*[[SYBR Gold]]		*[[SYBR Gold]]
		+
		+	===Equipment===
		+	*Electrophoresis apparatus

	==Procedure==		==Procedure==
-	===~~Make up~~ BPTE electrophoresis buffer===	+	===Prepare RNase free water===
		+	#Add DEPC to final concentration of 0.1% to H<sub>2</sub>O
		+	#Incubate 1hr at 37°C.
		+	#Autoclave for 15 mins at 15 psi.
		+
		+	===Prepare BPTE electrophoresis buffer===
	#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O		#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
	#*3 g of PIPES (free acid)		#*3 g of PIPES (free acid)
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	#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C		#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
	#Autoclave.		#Autoclave.
		+
		+	===Prepare glyoxal reaction mixture===
		+	#6mL DMSO
		+	#2mL deionized glyoxal
		+	#1.2mL of 10X BPTE electrophoresis buffer
		+	#0.6mL of 80% glycerol
		+
		+	Divide into small aliquots and store at -70°C.

	==Notes==		==Notes==

Reshma P. Shetty at 21:18, 5 December 2006

2006-12-05T21:18:51Z

←Older revision		Revision as of 21:18, 5 December 2006
Line 17:		Line 17:
	**0.6mL of 80% glycerol		**0.6mL of 80% glycerol
	*RNA gel loading buffer		*RNA gel loading buffer
		+	*RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
		+	**Add DEPC to final concentration of 0.1%.
		+	**Incubate 1hr at 37°C.
		+	**Autoclave for 15 mins at 15 psi.
	*[[SYBR Gold]]		*[[SYBR Gold]]

Reshma P. Shetty at 21:00, 5 December 2006

2006-12-05T21:00:25Z

←Older revision		Revision as of 21:00, 5 December 2006
Line 4:		Line 4:

	==Materials==		==Materials==
-	*[[10X BPTE electrophoresis buffer]]	+	*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
-	*~~[[SYBR Gold]]~~	+	**100 mM PIPES
		+	**300 mM Bis-Tris
		+	**10 mM EDTA
	*HPLC grade or better DMSO		*HPLC grade or better DMSO
	*Glyoxal		*Glyoxal
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	**1.2mL of 10X BPTE electrophoresis buffer		**1.2mL of 10X BPTE electrophoresis buffer
	**0.6mL of 80% glycerol		**0.6mL of 80% glycerol
		+	*RNA gel loading buffer
		+	*[[SYBR Gold]]

	==Procedure==		==Procedure==
		+	===Make up BPTE electrophoresis buffer===
		+	#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
		+	#*3 g of PIPES (free acid)
		+	#*6 g of Bis-Tris (free base)
		+	#*2 ml of 0.5 M EDTA
		+	#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
		+	#Autoclave.

	==Notes==		==Notes==
Line 21:		Line 32:
	==References==		==References==
	<biblio>		<biblio>
-	#MolecularCloning [[Molecular Cloning]]	+	#MolecularCloning [[doi:10.1101/pdb.prot4057\|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]]
	</biblio>		</biblio>