Endy:Northern blot, AlkPhos end-labeled probes - Revision history

Barry Canton: /* Hybridization (2 hours + overnight + >1 hour) */

2008-07-31T15:46:21Z

Hybridization (2 hours + overnight + >1 hour)

Felix Moser: /* Transfer of RNA onto Membrane by Turboblotter Capillary Transfer (3-4 hours) */

2008-07-24T14:22:32Z

Transfer of RNA onto Membrane by Turboblotter Capillary Transfer (3-4 hours)

←Older revision		Revision as of 14:22, 24 July 2008
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	===Transfer of RNA onto Membrane by Turboblotter Capillary Transfer (3-4 hours)===		===Transfer of RNA onto Membrane by Turboblotter Capillary Transfer (3-4 hours)===

-	''NOTE: Refer to Fig. 1 when setting up the TurboBlotter System.''	+	''NOTE: Refer to Fig. 1 when setting up the [http://www.whatman.com/TurboBlotter.asp TurboBlotter System].''

	#Place stack tray of transfer device on bench, making sure it is level.		#Place stack tray of transfer device on bench, making sure it is level.

Felix Moser: /* Prepare the Membrane (5-15 minutes) */

2008-07-24T14:21:22Z

Prepare the Membrane (5-15 minutes)

←Older revision		Revision as of 14:21, 24 July 2008
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	''NOTE: Be gentle with the membrane. The number of times a membrane can be stripped and re-probed is usually limited by physical damage to the blot.''		''NOTE: Be gentle with the membrane. The number of times a membrane can be stripped and re-probed is usually limited by physical damage to the blot.''

-	#Cut a piece of membrane to the dimensions of the agarose gel. Max dimensions for hybridization in 50mL tubes: 8 x 9 cm (circumference x diameter). If membrane is larger, sandwich between sheets of nylon mesh to allow buffer to penetrate overlap.	+	#Cut a piece of the [http://www.whatman.com/NytranNylonMembranes.aspx Nytran Supercharge membrane] to the dimensions of the agarose gel. Max dimensions for hybridization in 50mL tubes: 8 x 9 cm (circumference x diameter). If membrane is larger, sandwich between sheets of nylon mesh to allow buffer to penetrate overlap.
	#Wet the membrane by carefully laying it on top of Milli-Q water in a shallow tray. (Do not immerse the Immobilon-Ny+ membrane in liquid on the first liquid exposure. If you wet both sides, air can become trapped in the pores and form bubbles.)		#Wet the membrane by carefully laying it on top of Milli-Q water in a shallow tray. (Do not immerse the Immobilon-Ny+ membrane in liquid on the first liquid exposure. If you wet both sides, air can become trapped in the pores and form bubbles.)
	#Agitate the tray gently once the membrane is wet to completely immerse the membrane.		#Agitate the tray gently once the membrane is wet to completely immerse the membrane.

Felix Moser: /* Running the Gel (2-6hrs, depending on size of gel) */

2008-07-10T18:25:00Z

Running the Gel (2-6hrs, depending on size of gel)

←Older revision		Revision as of 18:25, 10 July 2008
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	#Soak gel 30 min in "Denaturing Buffer" (0.5 M NaOH/1.5 M NaCl (~400 mL)). Agitate occasionally.		#Soak gel 30 min in "Denaturing Buffer" (0.5 M NaOH/1.5 M NaCl (~400 mL)). Agitate occasionally.
	#Rinse the gel in ddH2O and Soak gel 30 min in "Neutralizing Buffer" (0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL))		#Rinse the gel in ddH2O and Soak gel 30 min in "Neutralizing Buffer" (0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL))
		+	#SOAK the gel in 20x SSC transfer buffer for 30min; shake slowly.
	''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''		''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''

Felix Moser: /* Running the Gel (2-6hrs, depending on size of gel) */

2008-07-10T18:04:52Z

Running the Gel (2-6hrs, depending on size of gel)

←Older revision		Revision as of 18:04, 10 July 2008
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	#Trim away areas of the gel to be stained with sybrGold. DO NOT trim the area BELOW the loading dye, since many RNA's run much lower than the dyes; trim the gel later once you know where all your RNA is. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in sarran wrap and store at 4°C until post-transfer gel is ready to be stained.		#Trim away areas of the gel to be stained with sybrGold. DO NOT trim the area BELOW the loading dye, since many RNA's run much lower than the dyes; trim the gel later once you know where all your RNA is. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in sarran wrap and store at 4°C until post-transfer gel is ready to be stained.
	#Soak gel 30 min in "Denaturing Buffer" (0.5 M NaOH/1.5 M NaCl (~400 mL)). Agitate occasionally.		#Soak gel 30 min in "Denaturing Buffer" (0.5 M NaOH/1.5 M NaCl (~400 mL)). Agitate occasionally.
-	#Rinse the gel in ddH2O and Soak gel 20 min in "Neutralizing Buffer" (0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL))	+	#Rinse the gel in ddH2O and Soak gel 30 min in "Neutralizing Buffer" (0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL))
	''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''		''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''

Felix Moser: /* Stain gel (can't do if continuing to northern) */

2008-07-09T20:48:24Z

Stain gel (can't do if continuing to northern)

←Older revision		Revision as of 20:48, 9 July 2008
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	''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''		''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels. These steps are the from the Turboblotter protocol''

-	===Stain gel ~~(can't do if continuing to northern)~~===	+	===Stain gel===
-	''If you ~~want to image~~ your RNA w/ dye as well as blot it for a Northern, it's a good idea simply to run a bigger gel w/ identical sample on one half of the gel for staining and the other half for the Northern.''	+	''If you stain your RNA w/ dye, you cannot use the same gel for the northern. If you want an RNA-stain image of the samples as well as blot it for a Northern, it's a good idea simply to run a bigger gel w/ identical sample on one half of the gel for staining and the other half for the Northern.''
	#Prepare fresh 1:10,000 dilution in RNase free water of SybrGold or SybrGreen (SybrGreen recommended by Sean, since it's specific for RNA; ppbly doesn't make a big difference, though).		#Prepare fresh 1:10,000 dilution in RNase free water of SybrGold or SybrGreen (SybrGreen recommended by Sean, since it's specific for RNA; ppbly doesn't make a big difference, though).
	#Ensure pH is 7.0-8.5.		#Ensure pH is 7.0-8.5.

Felix Moser: /* Stain gel (can't do if continuing to northern) */

2008-07-09T20:43:50Z

Stain gel (can't do if continuing to northern)

←Older revision		Revision as of 20:43, 9 July 2008
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	===Stain gel (can't do if continuing to northern)===		===Stain gel (can't do if continuing to northern)===
		+	''If you want to image your RNA w/ dye as well as blot it for a Northern, it's a good idea simply to run a bigger gel w/ identical sample on one half of the gel for staining and the other half for the Northern.''
	#Prepare fresh 1:10,000 dilution in RNase free water of SybrGold or SybrGreen (SybrGreen recommended by Sean, since it's specific for RNA; ppbly doesn't make a big difference, though).		#Prepare fresh 1:10,000 dilution in RNase free water of SybrGold or SybrGreen (SybrGreen recommended by Sean, since it's specific for RNA; ppbly doesn't make a big difference, though).
	#Ensure pH is 7.0-8.5.		#Ensure pH is 7.0-8.5.

Felix Moser: /* "Neutralizing" Buffer */

2008-07-09T19:10:36Z

"Neutralizing" Buffer

←Older revision		Revision as of 19:10, 9 July 2008
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	==="Neutralizing" Buffer===		==="Neutralizing" Buffer===
-	*0.5M Tris-HCl pH 7	+	*0.5M Tris-HCl pH 7.4
	*1.5M NaCl		*1.5M NaCl
	*For 1L: 60.56g Tris, 87.66g NaCl, 800ml ddH2O, adjust pH w/ conc. HCl and bring volume to 1L w/ ddH2O.		*For 1L: 60.56g Tris, 87.66g NaCl, 800ml ddH2O, adjust pH w/ conc. HCl and bring volume to 1L w/ ddH2O.

Felix Moser: /* Hybridization (2 hours + overnight + >1 hour) */

2008-07-07T19:57:58Z

Hybridization (2 hours + overnight + >1 hour)

←Older revision		Revision as of 19:57, 7 July 2008
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	'''Hybridization and Wash:'''		'''Hybridization and Wash:'''
	# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL Falcon tube, RNA facing in. Make sure the blot doesn't overlap itself. 50 mL conical tubes fit in the Sauer Lab "Bambino" mini-hybridization oven. If blot is too large for a 50 mL Falcon tube, use large glass tubes and Baker Lab hybridization oven.		# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL Falcon tube, RNA facing in. Make sure the blot doesn't overlap itself. 50 mL conical tubes fit in the Sauer Lab "Bambino" mini-hybridization oven. If blot is too large for a 50 mL Falcon tube, use large glass tubes and Baker Lab hybridization oven.
-	# Pre-heat the ~~rquired~~ volume of prepared hybridization buffer to 55*C. The volume of buffer should be equivalent to 0.25ml/cm2 of membrane; this may be reduced to half that volume for large blots hybridized in plastic bags or in bottles. (For our 9x9cm blot in the 50ml Falcon tube, we used 10ml of hybridization buffer).	+	# Pre-heat the required volume of prepared hybridization buffer to 55*C. The volume of buffer should be equivalent to 0.25ml/cm2 of membrane; this may be reduced to half that volume for large blots hybridized in plastic bags or in bottles. (For our 9x9cm blot in the 50ml Falcon tube, we used 10ml of hybridization buffer).
-	#Place the blot into the hybridization buffer and ~~prehybridize~~ for @ ~~lest~~ 15 min @ 55*C in a shaking water bath or hybridization oven. We used the Sauer Lab's "Bambino" hybridization oven.	+	#Place the blot into the hybridization buffer and pre-hybridize for @ least 15 min @ 55*C in a shaking water bath or hybridization oven. We used the Sauer Lab's "Bambino" hybridization oven.
-	#Add the ~~lableled~~ probe to the buffer used for the pre-hybridization step. Typically use 5-10ng/ml. Avoid placing the probe directly on the blot. Try taking out a small aliquot of buffer and mixing that with the probe before returning the mixture to the bulk of the hybridization buffer.	+	#Add the labeled probe to the buffer used for the pre-hybridization step. Typically use 5-10ng/ml. Avoid placing the probe directly on the blot. Try taking out a small aliquot of buffer and mixing that with the probe before returning the mixture to the bulk of the hybridization buffer.
	#Hybridize @ 55C overnight. Stringency can be adjusted by altering the hybridization temp between 50C and 75*C.		#Hybridize @ 55C overnight. Stringency can be adjusted by altering the hybridization temp between 50C and 75*C.
	#Preheat the primary wash buffer to 55*C.		#Preheat the primary wash buffer to 55*C.

Felix Moser: /* Hybridization (2 hours + overnight + >1 hour) */

2008-07-02T19:01:12Z

Hybridization (2 hours + overnight + >1 hour)

←Older revision		Revision as of 19:01, 2 July 2008
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	#Repeat wash with secondary wash buffer for 5min @ room temp.		#Repeat wash with secondary wash buffer for 5min @ room temp.
	#Drain excess wash buffer from the blot and place the blot face-up on a clean, non-absorbent flat surface. DO NOT allow blot to dry.		#Drain excess wash buffer from the blot and place the blot face-up on a clean, non-absorbent flat surface. DO NOT allow blot to dry.
-	'''Detection with ECF substrate:'''	+	'''Detection with ECF substrate:'''<br>
	''ECF is widely preferred as a fluorescent detection agent for this assay, based on people we talked to in the Baker lab ~~Felix Moser''		''ECF is widely preferred as a fluorescent detection agent for this assay, based on people we talked to in the Baker lab ~~Felix Moser''
	#Pour entire contents of the bottle containing the detection buffer into the bottle which contains the ECF detection reagent. Shake the bottle gently for about 10min to fully dissolve the ECF substrate.		#Pour entire contents of the bottle containing the detection buffer into the bottle which contains the ECF detection reagent. Shake the bottle gently for about 10min to fully dissolve the ECF substrate.

←Older revision		Revision as of 15:46, 31 July 2008
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	===Hybridization (2 hours + overnight + >1 hour)===		===Hybridization (2 hours + overnight + >1 hour)===
-	''This part highly depends on the type of labeling one is doing; protocols vary widely in buffers used, types of probe preparation, and hybridization protocol. We used the GE Healthcare ~~AlkaPhos~~ kit; the protocol below is essentially copy-pasted from the kit spec sheet.''	+	''This part highly depends on the type of labeling one is doing; protocols vary widely in buffers used, types of probe preparation, and hybridization protocol. We used the GE Healthcare AlkPhos kit; the protocol below is essentially copy-pasted from the kit spec sheet.''
	<br>'''Need the following solutions:'''		<br>'''Need the following solutions:'''
	*Hybridization Buffer		*Hybridization Buffer
Line 148:		Line 148:
	'''Hybridization and Wash:'''		'''Hybridization and Wash:'''
	# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL Falcon tube, RNA facing in. Make sure the blot doesn't overlap itself. 50 mL conical tubes fit in the Sauer Lab "Bambino" mini-hybridization oven. If blot is too large for a 50 mL Falcon tube, use large glass tubes and Baker Lab hybridization oven.		# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL Falcon tube, RNA facing in. Make sure the blot doesn't overlap itself. 50 mL conical tubes fit in the Sauer Lab "Bambino" mini-hybridization oven. If blot is too large for a 50 mL Falcon tube, use large glass tubes and Baker Lab hybridization oven.
-	# Pre-heat the required volume of prepared hybridization buffer to ~~55*C~~. The volume of buffer should be equivalent to 0.25ml/cm2 of membrane; this may be reduced to half that volume for large blots hybridized in plastic bags or in bottles. (For our 9x9cm blot in the 50ml Falcon tube, we used 10ml of hybridization buffer).	+	# Pre-heat the required volume of prepared hybridization buffer to 30C. The volume of buffer should be equivalent to 0.25ml/cm2 of membrane; this may be reduced to half that volume for large blots hybridized in plastic bags or in bottles. (For our 9x9cm blot in the 50ml Falcon tube, we used 10ml of hybridization buffer).
-	#Place the blot into the hybridization buffer and pre-hybridize for @ least 15 min @ ~~55*C~~ in a shaking water bath or hybridization oven. We used the Sauer Lab's "Bambino" hybridization oven.	+	#Place the blot into the hybridization buffer and pre-hybridize for @ least 15 min @ 30C in a shaking water bath or hybridization oven. We used the Sauer Lab's "Bambino" hybridization oven.
	#Add the labeled probe to the buffer used for the pre-hybridization step. Typically use 5-10ng/ml. Avoid placing the probe directly on the blot. Try taking out a small aliquot of buffer and mixing that with the probe before returning the mixture to the bulk of the hybridization buffer.		#Add the labeled probe to the buffer used for the pre-hybridization step. Typically use 5-10ng/ml. Avoid placing the probe directly on the blot. Try taking out a small aliquot of buffer and mixing that with the probe before returning the mixture to the bulk of the hybridization buffer.
-	#Hybridize @ ~~55C~~ overnight. Stringency can be adjusted by altering the hybridization temp between 50C and 75*C.	+	#Hybridize @ 30C overnight. Stringency can be adjusted by altering the hybridization temp between 50C and 75C. We're using 30C cause our probes are 17nt long.
-	#Preheat the primary wash buffer to ~~55*C~~.	+	#Preheat the primary wash buffer to 30C.
-	#Transfer the blot to the an excess of primary wash buffer and wash for 10min @ ~~55*C~~, with gentle agitation.	+	#Transfer the blot to the an excess of primary wash buffer and wash for 10min @ 30C, with gentle agitation.
-	#Repeat wash with primary wash buffer for 10min @~~55*C~~.	+	#Repeat wash with primary wash buffer for 10min @30C.
	#Transfer blot to new container and wash with secondary wash buffer for 5 min @ room temp.		#Transfer blot to new container and wash with secondary wash buffer for 5 min @ room temp.
	#Repeat wash with secondary wash buffer for 5min @ room temp.		#Repeat wash with secondary wash buffer for 5min @ room temp.