Knight:RNA electrophoresis/Native - Revision history

Reshma P. Shetty at 01:22, 19 July 2007

Reshma P. Shetty — Thu, 19 Jul 2007 01:22:15 GMT

←Older revision		Revision as of 01:22, 19 July 2007
Line 1:		Line 1:
	==Overview==		==Overview==
-	Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure.	+	Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure. Since this is a native gel you are more likely to see a smear of RNA and/or multiple bands because of RNA secondary structure.

	==Procedure==		==Procedure==
Line 33:		Line 33:
	#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA]		#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA]
	#Fermentas [http://www.fermentas.com/catalog/electrophoresis/rnaguide.htm Introduction: RNA Electrophoresis]		#Fermentas [http://www.fermentas.com/catalog/electrophoresis/rnaguide.htm Introduction: RNA Electrophoresis]
		+
	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:In vitro]]		[[Category:In vitro]]
	[[Category:RNA]]		[[Category:RNA]]

Reshma P. Shetty at 18:48, 14 February 2007

Reshma P. Shetty — Wed, 14 Feb 2007 18:48:16 GMT

←Older revision		Revision as of 18:48, 14 February 2007
Line 1:		Line 1:
-	~~<font color=red>Protocol in progress. Use at your own risk.</font>~~
-
	==Overview==		==Overview==
	Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure.		Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure.
Line 9:		Line 7:
	#Generally load 1 μg and 2.5 μg samples.		#Generally load 1 μg and 2.5 μg samples.
	#Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?		#Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?
-	#*''TAE buffer also ~~works but TBE is likely preferable~~.''	+	#*''TAE buffer also seems to work equally well in my hands for ~1000bp transcripts.''
	#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.		#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
	#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).		#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
Line 21:		Line 19:

	===Staining===		===Staining===
-	#Dilute SYBR ~~Green II~~ 10,000-fold into 1X running buffer.	+	#Dilute SYBR Gold 10,000-fold into 1X running buffer.
	#*Note that SYBR gold is preferable to SYBR green II		#*Note that SYBR gold is preferable to SYBR green II
	#Incubate agarose gel in staining solution for 40 minutes.		#Incubate agarose gel in staining solution for 40 minutes.

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Wed, 13 Dec 2006 01:50:01 GMT

Procedure

←Older revision		Revision as of 01:50, 13 December 2006
Line 9:		Line 9:
	#Generally load 1 μg and 2.5 μg samples.		#Generally load 1 μg and 2.5 μg samples.
	#Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?		#Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?
		+	#*''TAE buffer also works but TBE is likely preferable.''
	#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.		#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
	#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).		#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).

Reshma P. Shetty at 01:49, 13 December 2006

Reshma P. Shetty — Wed, 13 Dec 2006 01:49:14 GMT

←Older revision		Revision as of 01:49, 13 December 2006
Line 11:		Line 11:
	#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.		#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
	#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).		#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
		+	#*''I tried ~65V total.''
	#Use an RNase free aliquot of [[Knight:Loading dye\|loading buffer]] (i.e. made with RNase free water).		#Use an RNase free aliquot of [[Knight:Loading dye\|loading buffer]] (i.e. made with RNase free water).
-	#Ladder	+	#RNA Ladder
	#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]		#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]
	#*2 μL loading dye		#*2 μL loading dye
	#*8 μL H<sub>2</sub>O		#*8 μL H<sub>2</sub>O
		+	#Also run a DNA ladder

	===Staining===		===Staining===
	#Dilute SYBR Green II 10,000-fold into 1X running buffer.		#Dilute SYBR Green II 10,000-fold into 1X running buffer.
-	#*Note that SYBR gold is preferable.	+	#*Note that SYBR gold is preferable to SYBR green II
-	#Incubate agarose gel in staining solution for ~~20-~~40 minutes.	+	#Incubate agarose gel in staining solution for 40 minutes.
	#Visualize on gel imager. (No destaining needed).		#Visualize on gel imager. (No destaining needed).

Reshma P. Shetty: /* Staining */

Reshma P. Shetty — Tue, 12 Dec 2006 22:14:15 GMT

Staining

←Older revision		Revision as of 22:14, 12 December 2006
Line 18:		Line 18:

	===Staining===		===Staining===
-	#Dilute SYBR Green II 10,000-fold into 1X running buffer.	+	#Dilute SYBR Green II 10,000-fold into 1X running buffer.
		+	#*Note that SYBR gold is preferable.
	#Incubate agarose gel in staining solution for 20-40 minutes.		#Incubate agarose gel in staining solution for 20-40 minutes.
-	#Visualize on gel imager.	+	#Visualize on gel imager. (No destaining needed).

	==Notes==		==Notes==

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Tue, 12 Dec 2006 22:13:43 GMT

Procedure

←Older revision		Revision as of 22:13, 12 December 2006
Line 11:		Line 11:
	#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.		#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
	#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).		#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
-	#Use an RNase free aliquot of loading buffer (i.e. made with RNase free water).	+	#Use an RNase free aliquot of [[Knight:Loading dye\|loading buffer]] (i.e. made with RNase free water).
	#Ladder		#Ladder
	#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]		#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]
	#*2 μL loading dye		#*2 μL loading dye
	#*8 μL H<sub>2</sub>O		#*8 μL H<sub>2</sub>O
		+
		+	===Staining===
		+	#Dilute SYBR Green II 10,000-fold into 1X running buffer.
		+	#Incubate agarose gel in staining solution for 20-40 minutes.
		+	#Visualize on gel imager.

	==Notes==		==Notes==

Reshma P. Shetty at 21:41, 12 December 2006

Reshma P. Shetty — Tue, 12 Dec 2006 21:41:08 GMT

←Older revision		Revision as of 21:41, 12 December 2006
Line 15:		Line 15:
	#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]		#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]
	#*2 μL loading dye		#*2 μL loading dye
-	#*8 μL H<sub>2</sub>	+	#*8 μL H<sub>2</sub>O

	==Notes==		==Notes==

Reshma P. Shetty at 21:40, 12 December 2006

Reshma P. Shetty — Tue, 12 Dec 2006 21:40:55 GMT

←Older revision		Revision as of 21:40, 12 December 2006
Line 11:		Line 11:
	#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.		#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
	#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).		#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
		+	#Use an RNase free aliquot of loading buffer (i.e. made with RNase free water).
		+	#Ladder
		+	#*2 μL [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]
		+	#*2 μL loading dye
		+	#*8 μL H<sub>2</sub>

	==Notes==		==Notes==

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Tue, 12 Dec 2006 16:41:00 GMT

Notes

←Older revision		Revision as of 16:41, 12 December 2006
Line 15:		Line 15:
	*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>		*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>
	*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>		*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>
-	*''TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 bp RNA and for denaturing polyacrylamide gel electrophoresis.'' <cite>Fermentas</cite>	+	*''TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 nt RNA and for denaturing polyacrylamide gel electrophoresis.'' <cite>Fermentas</cite>

	==References==		==References==

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Tue, 12 Dec 2006 16:40:41 GMT

Notes

←Older revision		Revision as of 16:40, 12 December 2006
Line 15:		Line 15:
	*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>		*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>
	*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>		*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>
-	*''TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 b RNA and for denaturing polyacrylamide gel electrophoresis.'' <cite>Fermentas</cite>	+	*''TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 bp RNA and for denaturing polyacrylamide gel electrophoresis.'' <cite>Fermentas</cite>

	==References==		==References==