Alm:Agarose gel electrophoresis - Revision history

ClarkeS: /* IBI (Shelton Scientific) 6x loading dye */

ClarkeS — Thu, 15 Jan 2009 16:02:43 GMT

IBI (Shelton Scientific) 6x loading dye

←Older revision		Revision as of 16:02, 15 January 2009
Line 48:		Line 48:
	15% Ficoll in a special TRIS Dye		15% Ficoll in a special TRIS Dye

-	Dye #1: Light blue slightly slower than Xylene Cyanol migrating at around 4,oo base pairs in a	+	Dye #1: Light blue slightly slower than Xylene Cyanol migrating at around 4,000 base pairs in a
	1% agarose gel.		1% agarose gel.

ClarkeS: /* Staining and visualization */

ClarkeS — Fri, 25 Jul 2008 19:24:36 GMT

Staining and visualization

←Older revision		Revision as of 19:24, 25 July 2008
Line 23:		Line 23:

	===Staining and visualization===		===Staining and visualization===
-
	*'''Wear blue/purple nitrile gloves when handling the items at the staining station and the gel imager.'''		*'''Wear blue/purple nitrile gloves when handling the items at the staining station and the gel imager.'''
-	#Place the gel in the plastic box.	+	#Place the gel in the plastic box in the hood.
-	#Stain for 1 hour in EtBr solution (concentration??).	+	#Stain for 2 minutes to 1 hour in EtBr solution (depending on the concentration). Cover the plastic box with the cardboard lid to prevent bleaching of the dye.
-	#Pour the EtBr back into the light-proof bottle.	+	#Pour the EtBr back into the light-proof bottle covered in aluminum foil.
-	#Destain with used TAE for 2 minutes.	+	#Destain with used TAE for 30 seconds to 2 minutes, rocking the box gently.
-	#Pour the TAE back into the bottle.	+	#Pour the TAE back into the bottle with the funnel.
-	#Image the gel on a UV light box or the computerized gel ~~imager~~ in the Polz lab.	+	#Image the gel on a UV light box or the computerized gel documentation (geldoc) system in the Thompson lab or Polz lab.
-	#Print out the image for your notebook, annotating the lanes, marker, and bands.	+	#Print out the image for your notebook, annotating the lanes, marker, and bands. Also note electrophoresis details like amount of DNA loaded, dye, voltage, time.
	#Note agreement with/variation from expected result.		#Note agreement with/variation from expected result.

ClarkeS: /* Notes */

ClarkeS — Fri, 25 Jul 2008 19:19:18 GMT

Notes

←Older revision		Revision as of 19:19, 25 July 2008
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	==Notes==		==Notes==
		+	===NEB Gel Loading Dye, Blue (6X)===
		+	is a pre-mixed loading buffer with one tracking dye for agarose and non-denaturing poylacrylamide gel electrophoresis. This solution contains SDS, which often results in sharper bands, as some restriction enzymes are known to remain bound to DNA following cleavage. EDTA is also included to chelate magnesium (up to 10 mM) in enzymatic reactions, thereby stopping the reaction. Bromophenol blue is the standard tracking dye for electrophoresis. It migrates at approximately 300 bp on a standard 1% TBE agarose gel.
		+
		+	1X Gel Loading Dye, Blue (6X):
		+	*2.5 % Ficoll 400
		+	*11 mM EDTA
		+	*3.3 mM Tris-HCl
		+	*0.017 % SDS
		+	*0.015 % Bromophenol Blue
		+	*pH 8.0 @ 25°C
		+
		+	===IBI (Shelton Scientific) 6x loading dye===
		+	15% Ficoll in a special TRIS Dye
		+
		+	Dye #1: Light blue slightly slower than Xylene Cyanol migrating at around 4,oo base pairs in a
		+	1% agarose gel.
		+
		+	Dye #2: Indigo dye migrates similar to Bromophenol Blue at around 600 base pair in a 1%
		+	agarose gel.
		+
		+	Dye #3: Magenta dye migrates at around 150 base pairs in a 1% agarose gel.

	==Contact==		==Contact==

ClarkeS: /* Electrophoresis */

ClarkeS — Thu, 20 Sep 2007 20:54:41 GMT

Electrophoresis

←Older revision		Revision as of 20:54, 20 September 2007
Line 19:		Line 19:
	#Add 6x loading dye to your samples.		#Add 6x loading dye to your samples.
	#Make sure the red electrode is at the opposite end from the wells. You can check the progress of the DNA with the loading dyes to check the direc		#Make sure the red electrode is at the opposite end from the wells. You can check the progress of the DNA with the loading dyes to check the direc
-	#Set the time and settings on the power supply by pressing Mode to change fields. (typical settings for DNA: 90V constant voltage, 45 minutes)	+	#Set the time and settings on the power supply by pressing Mode to change fields. (typical settings for 100s bp fragments of DNA: 90V constant voltage, 45 minutes)
	#Slide on the lid and start the power supply.		#Slide on the lid and start the power supply.

ClarkeS: /* Materials */

ClarkeS — Thu, 20 Sep 2007 20:53:47 GMT

Materials

←Older revision		Revision as of 20:53, 20 September 2007
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	==Materials==		==Materials==

-	*6X loading dye	+	*[[Agarose_gel_loading_dye\|6X loading dye]]
	*[[TAE]] + agarose 1%		*[[TAE]] + agarose 1%
	*1X [[TAE]] (~200ml)		*1X [[TAE]] (~200ml)

ClarkeS: Alm:Gel electrophoresis moved to Alm:Agarose gel electrophoresis

ClarkeS — Thu, 20 Sep 2007 20:52:37 GMT

Alm:Gel electrophoresis moved to Alm:Agarose gel electrophoresis

←Older revision

Revision as of 20:52, 20 September 2007

ClarkeS: /* References */

ClarkeS — Thu, 20 Sep 2007 20:52:22 GMT

References

←Older revision		Revision as of 20:52, 20 September 2007
Line 42:		Line 42:
	==References==		==References==
	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->		<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
-	~~<biblio>~~	+	*[[Agarose_gel_electrophoresis]]
-	~~</biblio>~~	+

	[[Category:Protocol]]		[[Category:Protocol]]

ClarkeS: /* Staining and visualization */

ClarkeS — Thu, 20 Sep 2007 20:50:04 GMT

Staining and visualization

←Older revision		Revision as of 20:50, 20 September 2007
Line 24:		Line 24:
	===Staining and visualization===		===Staining and visualization===

-	*'''Wear blue/purple nitrile gloves when handling the items at the staining station.'''	+	*'''Wear blue/purple nitrile gloves when handling the items at the staining station and the gel imager.'''
	#Place the gel in the plastic box.		#Place the gel in the plastic box.
	#Stain for 1 hour in EtBr solution (concentration??).		#Stain for 1 hour in EtBr solution (concentration??).

ClarkeS: /* Materials */

ClarkeS — Thu, 20 Sep 2007 20:49:31 GMT

Materials

←Older revision		Revision as of 20:49, 20 September 2007
Line 5:		Line 5:

	*6X loading dye		*6X loading dye
-	*[[TAE]] agarose 1%	+	*[[TAE]] + agarose 1%
-	*1X TAE (~200ml)	+	*1X [[TAE]] (~200ml)
-	*gel box (casting tray optional)	+	*gel box (casting tray optional) and power supply
-	*power supply	+	*[[Ethidium Bromide]] staining solution
-	*[[Ethidium ~~bromide~~]] staining solution	+
	*used TAE destaining solution		*used TAE destaining solution
	*UV box/gel imager		*UV box/gel imager

ClarkeS at 20:48, 20 September 2007

ClarkeS — Thu, 20 Sep 2007 20:48:09 GMT

←Older revision		Revision as of 20:48, 20 September 2007
Line 1:		Line 1:
-	Electrophoresis	+	==Overview==
		+	How to use gel electrophoresis to separate, measure and visualize DNA pieces.
		+
		+	==Materials==
		+
		+	*6X loading dye
		+	*[[TAE]] agarose 1%
		+	*1X TAE (~200ml)
		+	*gel box (casting tray optional)
		+	*power supply
		+	*[[Ethidium bromide]] staining solution
		+	*used TAE destaining solution
		+	*UV box/gel imager
		+
		+	==Procedure==
		+
		+	===Electrophoresis===

	#Microwave TAE agarose 1% to melt. Pour 30 ml for a small gel into a small flask/beaker to cool a bit, then pour into the gel box with the correct comb for your size/number of samples.		#Microwave TAE agarose 1% to melt. Pour 30 ml for a small gel into a small flask/beaker to cool a bit, then pour into the gel box with the correct comb for your size/number of samples.
Line 7:		Line 23:
	#Slide on the lid and start the power supply.		#Slide on the lid and start the power supply.

-	Staining and visualization	+	===Staining and visualization===
-		+
-	~~'''Wear blue/purple nitrile gloves when handling the items at the staining station.'''~~	+

		+	*'''Wear blue/purple nitrile gloves when handling the items at the staining station.'''
	#Place the gel in the plastic box.		#Place the gel in the plastic box.
	#Stain for 1 hour in EtBr solution (concentration??).		#Stain for 1 hour in EtBr solution (concentration??).
Line 19:		Line 34:
	#Print out the image for your notebook, annotating the lanes, marker, and bands.		#Print out the image for your notebook, annotating the lanes, marker, and bands.
	#Note agreement with/variation from expected result.		#Note agreement with/variation from expected result.
		+
		+	==Notes==
		+
		+	==Contact==
		+	*Arne Materna
		+	*Sean Clarke
		+
		+	==References==
		+	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
		+	<biblio>
		+	</biblio>
		+
		+	[[Category:Protocol]]
		+
		+	[[Category:In vitro]]
		+
		+	[[Category:DNA]]
		+
		+	[[Category:RNA]]
		+
		+	[[Category:Protein]]
		+
		+	[[Category:Alm]]