RNA electrophoresis - Revision history

Jakob Suckale: /* RNA size markers / ladders */

2009-03-02T14:33:10Z

RNA size markers / ladders

←Older revision		Revision as of 14:33, 2 March 2009
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	* [http://www.fermentas.com/catalog/electrophoresis/rnaladders.htm RiboRuler RNA ladders from Fermentas $130/145 25 lanes]		* [http://www.fermentas.com/catalog/electrophoresis/rnaladders.htm RiboRuler RNA ladders from Fermentas $130/145 25 lanes]
	* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=15623200&messageType=catProductDetail Invitrogen 0.5-10kb RNA ladder €150 75µg]		* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=15623200&messageType=catProductDetail Invitrogen 0.5-10kb RNA ladder €150 75µg]
-	* [https://www.roche-applied-science.com/servlet/~~RCConfigureUser~~?~~URL~~=~~StoreFramesetView&storeId=10201&catalogId=10201&langId=-~~3~~&countryId=de~~ Roche DIG-labelled RNA ladder ~~€150~~ 2µg ~~- €260 4µg~~]	+	* [https://www.roche-applied-science.com/servlet/RCProductDisplay?productId=3.6.12.4.5.1 Roche DIG-labelled RNA ladder €160 2µg]
	* [http://www.protocol-online.org/biology-forums/posts/12166.html short discussion on using DNA as size marker for RNA gels]		* [http://www.protocol-online.org/biology-forums/posts/12166.html short discussion on using DNA as size marker for RNA gels]

Jakob Suckale: RNA ladders

2009-01-30T17:28:07Z

RNA ladders

←Older revision		Revision as of 17:28, 30 January 2009
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	*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. <cite>MolecularCloning1</cite>		*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. <cite>MolecularCloning1</cite>

-	==~~Acknowledgments~~==	+	==RNA size markers / ladders==
		+	* [http://www.neb.com/nebecomm/products/productN0362.asp NEB ssRNA ladder $58], [http://www.neb.com/nebecomm/products/productN0363.asp dsRNA ladder $75, 25 lanes]
		+	* [http://www.fermentas.com/catalog/electrophoresis/rnaladders.htm RiboRuler RNA ladders from Fermentas $130/145 25 lanes]
		+	* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=15623200&messageType=catProductDetail Invitrogen 0.5-10kb RNA ladder €150 75µg]
		+	* [https://www.roche-applied-science.com/servlet/RCConfigureUser?URL=StoreFramesetView&storeId=10201&catalogId=10201&langId=-3&countryId=de Roche DIG-labelled RNA ladder €150 2µg - €260 4µg]
		+	* [http://www.protocol-online.org/biology-forums/posts/12166.html short discussion on using DNA as size marker for RNA gels]

	==Specific Protocols==		==Specific Protocols==

Jakob Suckale: specific protocols

2009-01-30T17:19:09Z

specific protocols

←Older revision		Revision as of 17:19, 30 January 2009
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	==Acknowledgments==		==Acknowledgments==

		+	==Specific Protocols==
		+	*[[Jacobs:Protocol RNA Agarose Gel]]
		+	*[[Knight:RNA electrophoresis/Native]]
		+	*[[Jacobs:Protocol Agarose Formaldehyde Gel Preparation for RNA Electrophoresis]]
		+	*[[Knight:RNA electrophoresis/Denaturing]]
		+	*[[Endy:Northern Blot, 32P End-Labeled Probes#Process the Gel (3-4 hours)]]

	==References==		==References==
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	#ProtocolsOnline [http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html RNA electrophoresis]		#ProtocolsOnline [http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html RNA electrophoresis]
	</biblio>		</biblio>
-
-	~~==Specific Protocols==~~
-	~~#[[Endy:Northern Blot, 32P End-Labeled Probes#Process the Gel (3-4 hours)]]~~
-	~~#[[Knight:RNA electrophoresis]]~~

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:RNA]]		[[Category:RNA]]
	[[Category:In vitro]]		[[Category:In vitro]]

Reshma P. Shetty at 16:52, 6 December 2006

2006-12-06T16:52:26Z

←Older revision		Revision as of 16:52, 6 December 2006
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	#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion		#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion
	#LabGuidetoRNA isbn=0-471-12536-9		#LabGuidetoRNA isbn=0-471-12536-9
		+	#ProtocolsOnline [http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html RNA electrophoresis]
	</biblio>		</biblio>

Reshma P. Shetty: /* Critical steps */

2006-12-06T16:37:57Z

Critical steps

←Older revision		Revision as of 16:37, 6 December 2006
Line 17:		Line 17:

	==Critical steps==		==Critical steps==
-	*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient.	+	*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient. <cite>LabGuidetoRNA</cite>
	*The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.		*The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.

Reshma P. Shetty: /* References */

2006-12-06T16:37:33Z

References

←Older revision		Revision as of 16:37, 6 December 2006
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	#CurrentProtocols [[doi:10.1002/0471142727.mb0409s67\|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]]		#CurrentProtocols [[doi:10.1002/0471142727.mb0409s67\|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]]
	#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion		#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion
		+	#LabGuidetoRNA isbn=0-471-12536-9
	</biblio>		</biblio>

Reshma P. Shetty at 16:36, 6 December 2006

2006-12-06T16:36:50Z

←Older revision		Revision as of 16:36, 6 December 2006
Line 17:		Line 17:

	==Critical steps==		==Critical steps==
-	*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions.	+	*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient.
	*The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.		*The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.

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	*For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. <cite>RNAmethodologies</cite>		*For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. <cite>RNAmethodologies</cite>
	*[[Tom Knight]] strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde.		*[[Tom Knight]] strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde.
-	*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this.	+	*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. <cite>MolecularCloning1</cite>

	==Acknowledgments==		==Acknowledgments==
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	#MolecularCloning2 [[doi:10.1101/pdb.prot4050\|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde]]		#MolecularCloning2 [[doi:10.1101/pdb.prot4050\|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde]]
	#RNAmethodologies isbn=0-12-249695-7		#RNAmethodologies isbn=0-12-249695-7
-	#CurrentProtocols [[doi:10.1002/0471142727.mb0409s67\|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]]	+	#CurrentProtocols [[doi:10.1002/0471142727.mb0409s67\|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]]
		+	#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion
	</biblio>		</biblio>

Reshma P. Shetty at 23:27, 5 December 2006

2006-12-05T23:27:38Z

←Older revision		Revision as of 23:27, 5 December 2006
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	==Troubleshooting==		==Troubleshooting==
-	*RNases are the biggest problem in RNA work. Use proper precautions.	+	*RNases are the biggest problem in RNA work. [[Avoiding RNase contamination\|Use proper precautions]].
-	*The electrophoresis apparatus can be contaminated with RNases. First, clean chamber, casting tray and combs with detergent solution. Soak all in solution of 3% H<sub>2</sub>O<sub>2</sub> for 10 mins at room temperature and then rinse with large amounts of DEPC treated, autoclaved H<sub>2</sub>O. (At a minimum, do this with the gel comb).	+

	==Notes==		==Notes==

Reshma P. Shetty: /* Troubleshooting */

2006-12-05T21:12:22Z

Troubleshooting

←Older revision		Revision as of 21:12, 5 December 2006
Line 22:		Line 22:
	==Troubleshooting==		==Troubleshooting==
	*RNases are the biggest problem in RNA work. Use proper precautions.		*RNases are the biggest problem in RNA work. Use proper precautions.
-	*The electrophoresis apparatus can be contaminated with RNases. ~~At a minimum~~, ~~soak comb~~ in solution of 3% H<sub>2</sub>O<sub>2</sub> ~~or 0.5% SDS, 50mM EDTA~~ and then rinse with large amounts of DEPC treated, autoclaved H<sub>2</sub>O.	+	*The electrophoresis apparatus can be contaminated with RNases. First, clean chamber, casting tray and combs with detergent solution. Soak all in solution of 3% H<sub>2</sub>O<sub>2</sub> for 10 mins at room temperature and then rinse with large amounts of DEPC treated, autoclaved H<sub>2</sub>O. (At a minimum, do this with the gel comb).

	==Notes==		==Notes==

Reshma P. Shetty: /* Troubleshooting */

2006-12-05T21:09:32Z

Troubleshooting

←Older revision		Revision as of 21:09, 5 December 2006
Line 22:		Line 22:
	==Troubleshooting==		==Troubleshooting==
	*RNases are the biggest problem in RNA work. Use proper precautions.		*RNases are the biggest problem in RNA work. Use proper precautions.
-	*The electrophoresis apparatus can be contaminated with RNases. At a minimum, soak comb in solution of 3% H<sub>2</sub>O<sub>2</sub> or 0.5% SDS, 50mM EDTA and then rinse with large amounts of autoclaved H<sub>2</sub>O.	+	*The electrophoresis apparatus can be contaminated with RNases. At a minimum, soak comb in solution of 3% H<sub>2</sub>O<sub>2</sub> or 0.5% SDS, 50mM EDTA and then rinse with large amounts of DEPC treated, autoclaved H<sub>2</sub>O.

	==Notes==		==Notes==