Endy:Northern Blot, 32P End-Labeled Probes - Revision history

Jakob Suckale: /* Northern Blot Solutions */ SSC SDS links

2009-06-15T12:44:24Z

Northern Blot Solutions: SSC SDS links

←Older revision		Revision as of 12:44, 15 June 2009
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	===Prewash Solution (0.1x SSC/ 0.1% SDS)===		===Prewash Solution (0.1x SSC/ 0.1% SDS)===
	50 mL Total Volume		50 mL Total Volume
-	#*250 µL of 20x SSC	+	#*250 µL of 20x [[SSC]]
-	#*500 µL of 10% SDS	+	#*500 µL of 10% [[SDS]]
	#*49.25 mL of nuclease-free H2O		#*49.25 mL of nuclease-free H2O

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	50 mL Total Volume		50 mL Total Volume
	#*5 mL of 100x Denhart's Solution		#*5 mL of 100x Denhart's Solution
-	#*15 mL of 20x SSC	+	#*15 mL of 20x [[SSC]]
-	#*0.5 mL of 10% SDS	+	#*0.5 mL of 10% [[SDS]]
	#*29.5 mL of Nuclease-free H2O		#*29.5 mL of Nuclease-free H2O

	===Stringency Wash (6x SSC/ 0.1% SDS)===		===Stringency Wash (6x SSC/ 0.1% SDS)===
	200 mL Total Volume		200 mL Total Volume
-	#*2 mL of 10% SDS	+	#*2 mL of 10% [[SDS]]
-	#*60 mL of 20X SSC	+	#*60 mL of 20X [[SSC]]
	#*138 mL of nuclease-free H2O		#*138 mL of nuclease-free H2O

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

Reshma P. Shetty at 15:36, 14 March 2007

2007-03-14T15:36:20Z

←Older revision		Revision as of 15:36, 14 March 2007
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	#*138 mL of nuclease-free H2O		#*138 mL of nuclease-free H2O

-	[[Category:~~RNA~~ Protocol]]	+	[[Category:Protocol]] [[Category:RNA]] [[Category:In vitro]]

Cconboy at 19:48, 30 May 2006

2006-05-30T19:48:52Z

←Older revision		Revision as of 19:48, 30 May 2006
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-	Adapted from: Parker Lab Protocols, Sauer Lab, ~~hkeller~~, Molecular Cloning, Current Protocols in Molecular Biology,	+	Adapted from: Parker Lab Protocols, [[Sauer Lab]], [[Heather Keller]], Molecular Cloning, Current Protocols in Molecular Biology, Schleicher & Schuell Bioscience Turboblotter, Millipore Immobilon-Ny+ Transfer Membrane.
-	Schleicher & Schuell Bioscience Turboblotter, Millipore Immobilon-Ny+ Transfer Membrane.	+
	Contact: [[Caitlin Conboy]]		Contact: [[Caitlin Conboy]]

Cconboy: Northern Blot, 32P End-Labeled Probes moved to Endy:Northern Blot, 32P End-Labeled Probes

2006-04-12T19:53:10Z

Northern Blot, 32P End-Labeled Probes moved to Endy:Northern Blot, 32P End-Labeled Probes

←Older revision

Revision as of 19:53, 12 April 2006

Cconboy at 19:50, 12 April 2006

2006-04-12T19:50:46Z

Kathmc at 03:03, 17 June 2005

2005-06-17T03:03:40Z

←Older revision		Revision as of 03:03, 17 June 2005
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	===Probe Preparation===		===Probe Preparation===
	#For DNA probes: Design and order synthetic oligos (https://catalog.invitrogen.com/)		#For DNA probes: Design and order synthetic oligos (https://catalog.invitrogen.com/)
-	# End-Label [[Probe Prep, 32P End-Labeled ~~Propes~~]]	+	# End-Label [[Probe Prep, 32P End-Labeled Probes]]

	===Northern===		===Northern===
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	===Prepare the Membrane (5-15 minutes)===		===Prepare the Membrane (5-15 minutes)===

-	''NOTE: Be gentle with the membrane. The number of times a membrane can be stripped and re-probed is usually limited by physical ~~dammage~~ 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 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.

Cconboy: /* Experimental Samples */

2005-04-28T22:05:44Z

Experimental Samples

←Older revision		Revision as of 22:05, 28 April 2005
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	===Experimental Samples===		===Experimental Samples===
	Total cellular RNA [[RNA Extraction]]		Total cellular RNA [[RNA Extraction]]
-	~10 ?g of total cellular RNA, would contain 10 fg -1 pg of rare RNA, ~300 pg of moderately abundant RNA. (''Molecular Cloning'')	+	~10 µg of total cellular RNA, would contain 10 fg -1 pg of rare RNA, ~300 pg of moderately abundant RNA. (''Molecular Cloning'')

	===In Vitro Standard Curve Samples===		===In Vitro Standard Curve Samples===

Cconboy: /* Process the Gel (3-4 hours) */

2005-04-28T22:05:19Z

Process the Gel (3-4 hours)

←Older revision		Revision as of 22:05, 28 April 2005
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	#Soak gel 30 min in 0.05 M NaOH/1.5 M NaCl (~400 mL) without agitation.		#Soak gel 30 min in 0.05 M NaOH/1.5 M NaCl (~400 mL) without agitation.
	#Soak gel 20 min in 0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL)		#Soak gel 20 min in 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.''	+	''7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels.''
-		+

	===Prepare the Membrane (5-15 minutes)===		===Prepare the Membrane (5-15 minutes)===

Cconboy: /* Northern Blot Solutions */

2005-04-28T22:04:40Z

Northern Blot Solutions

←Older revision		Revision as of 22:04, 28 April 2005
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	#*5 g of Ficoll 400		#*5 g of Ficoll 400
	#*5 g of polyvinylpyrrolidone (Not polyvinylpolypyrrolidone!)		#*5 g of polyvinylpyrrolidone (Not polyvinylpolypyrrolidone!)
-	#*5 g of BSA Fraction V (stored at ~~4�C~~)	+	#*5 g of BSA Fraction V (stored at 4°C)
	#dissolve to 250 mL in nuclease-free H2O		#dissolve to 250 mL in nuclease-free H2O
	#Sterile filter and freeze in 5 mL aliquots		#Sterile filter and freeze in 5 mL aliquots
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	===Pre-hybridization Buffer===		===Pre-hybridization Buffer===
	50 mL Total Volume		50 mL Total Volume
-	#*5 mL of 100x ~~Denhart�s~~ Solution	+	#*5 mL of 100x Denhart's Solution
	#*15 mL of 20x SSC		#*15 mL of 20x SSC
	#*0.5 mL of 10% SDS		#*0.5 mL of 10% SDS

Cconboy at 22:03, 28 April 2005

2005-04-28T22:03:56Z

←Older revision		Revision as of 19:50, 12 April 2006
Line 1:		Line 1:
-	Adapted from: ~~cmc,~~ Parker Lab Protocols, Sauer Lab, hkeller, Molecular Cloning, Current Protocols in Molecular Biology,	+	Adapted from: Parker Lab Protocols, Sauer Lab, hkeller, Molecular Cloning, Current Protocols in Molecular Biology,
-	Schleicher & Schuell Bioscience Turboblotter, Millipore Immobilon-Ny+ Transfer Membrane	+	Schleicher & Schuell Bioscience Turboblotter, Millipore Immobilon-Ny+ Transfer Membrane.
		+	Contact: [[Caitlin Conboy]]

	==Samples to be probed==		==Samples to be probed==
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	#Cover the membrane with agarose gel, cut the gel to the size of the membrane, making sure there are no air bubbles between the gel and the membrane.		#Cover the membrane with agarose gel, cut the gel to the size of the membrane, making sure there are no air bubbles between the gel and the membrane.
	#Wet the top surface of the gel with transfer buffer and place 3 sheets of GB002 Blotting Paper, presoaked in transfer buffer on top of the gel.		#Wet the top surface of the gel with transfer buffer and place 3 sheets of GB002 Blotting Paper, presoaked in transfer buffer on top of the gel.
-	#Attach the ~~�buffer tray�~~ of the transfer device to the bottom tray using the circular alignment buttons to align both trays.	+	#Attach the buffer tray of the transfer device to the bottom tray using the circular alignment buttons to align both trays.
	#Fill the buffer tray with 125 ml transfer buffer for 7 x 8 cm to 11 x 14 cm transfers; (200 ml for 12 x 21 cm to 20 x 25 cm transfers).		#Fill the buffer tray with 125 ml transfer buffer for 7 x 8 cm to 11 x 14 cm transfers; (200 ml for 12 x 21 cm to 20 x 25 cm transfers).
-	#Start the transfer by connecting the gel stack with the buffer tray using the precut ~~�buffer wick�~~ (included in each blotter stack), presoaked in transfer buffer. Place the ~~�wick cover�~~ on top of the stack to prevent evaporation. Make sure the edges of the wick are immersed in the transfer buffer.	+	#Start the transfer by connecting the gel stack with the buffer tray using the precut buffer wick (included in each blotter stack), presoaked in transfer buffer. Place the wick cover on top of the stack to prevent evaporation. Make sure the edges of the wick are immersed in the transfer buffer.
	#Continue the transfer for 3 hr. Additional transfer time may be required for gels thicker than 4 mm or larger-size nucleic acids. ''(Try 4 hr, since gel is 1.5%, cmc 3.25.05)''		#Continue the transfer for 3 hr. Additional transfer time may be required for gels thicker than 4 mm or larger-size nucleic acids. ''(Try 4 hr, since gel is 1.5%, cmc 3.25.05)''
	#Disassembly: mark edges of gel and lane borders onto blot with pencil.		#Disassembly: mark edges of gel and lane borders onto blot with pencil.
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	===Hybridization (2 hours + overnight + >1 hour)===		===Hybridization (2 hours + overnight + >1 hour)===
-	# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL conical 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 conical 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 conical 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 conical tube, use large glass tubes and Baker Lab hybridization oven.
-	#Prewash Blot in 0.1x SSC/ 0.1% SDS for 1 hour at 65°C in hybridization oven. 10 - 15 mL of solution is required to cover the blot in a 50 mL tube, and sufficient for all steps. This incubation can be cut to 30 min if pressed ( says Sean.)	+	#Prewash Blot in 0.1x SSC/ 0.1% SDS for 1 hour at 65°C in hybridization oven. 10 - 15 mL of solution is required to cover the blot in a 50 mL tube, and sufficient for all steps. This incubation can be cut to 30 min if pressed (says [[Sean Moore\|Sean]].)
	#Remove Prewash		#Remove Prewash
-	#Prehybridize blot for > 1 hour in 10 - 15 mL of pre-hybridization solution at hybridization temp. Hybridization temp ~15°C below estimated Tm of probe. If reusing probe in hybridization solution, thaw probe as balance. ''NOTE: It is possible to store blots in prehybridization solution sort-term to indefinitely at 4°C or -20°C (Sean)''	+	#Prehybridize blot for > 1 hour in 10 - 15 mL of pre-hybridization solution at hybridization temp. Hybridization temp ~15°C below estimated Tm of probe. If reusing probe in hybridization solution, thaw probe as balance. ''NOTE: It is possible to store blots in prehybridization solution sort-term to indefinitely at 4°C or -20°C ([[Sean Moore\|Sean]])''
	#Remove prehybridization buffer if reusing probe, otherwise retain same buffer.		#Remove prehybridization buffer if reusing probe, otherwise retain same buffer.
	#Add hybridization buffer with old probe to blot or add new probe to pre-hybridization buffer. Hybridize >6 hrs at hybridization temp.		#Add hybridization buffer with old probe to blot or add new probe to pre-hybridization buffer. Hybridize >6 hrs at hybridization temp.

←Older revision		Revision as of 22:03, 28 April 2005
Line 40:		Line 40:

	#Set up the glyoxal denaturation reaction by combining 1-2 ul of RNA (up to 10 ug) with 10 ul of glyoxal reaction mixture. Run molecular weight markers for staining with sybrGold.		#Set up the glyoxal denaturation reaction by combining 1-2 ul of RNA (up to 10 ug) with 10 ul of glyoxal reaction mixture. Run molecular weight markers for staining with sybrGold.
-	#Incubate the RNA solutions for 60 minutes at ~~55�C~~. Chill the samples for 10 minutes in ice water and then centrifuge them for 5 seconds to deposit all of the fluid in the bottom of the microfuge tubes.	+	#Incubate the RNA solutions for 60 minutes at 55°C. Chill the samples for 10 minutes in ice water and then centrifuge them for 5 seconds to deposit all of the fluid in the bottom of the microfuge tubes.
	#While the samples are incubating, clean electrophoresis tank if necessary, and pour a 1.5% agarose gel in 1X BPTE (1.05 g agarose in 70 mL buffer). When set, cover the gel with sufficient buffer.		#While the samples are incubating, clean electrophoresis tank if necessary, and pour a 1.5% agarose gel in 1X BPTE (1.05 g agarose in 70 mL buffer). When set, cover the gel with sufficient buffer.
	#Add 2.5 ul of 6X gel loading buffer to the glyoxylated RNA samples, and without delay, load the glyoxylated RNA samples into the wells of the gel.		#Add 2.5 ul of 6X gel loading buffer to the glyoxylated RNA samples, and without delay, load the glyoxylated RNA samples into the wells of the gel.
	#Carry out electrophoresis at 70 Volts.		#Carry out electrophoresis at 70 Volts.
-	#Trim away areas of the gel to be stained with sybrGold. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in saran 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. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in saran wrap and store at 4°C until post-transfer gel is ready to be stained.
	#Soak gel 30 min in 0.05 M NaOH/1.5 M NaCl (~400 mL) without agitation.		#Soak gel 30 min in 0.05 M NaOH/1.5 M NaCl (~400 mL) without agitation.
	#Soak gel 20 min in 0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL)		#Soak gel 20 min in 0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL)
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	#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 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.
	#Transfer the membrane to a second tray containing transfer buffer (20 x SSC).		#Transfer the membrane to a second tray containing transfer buffer (20 x SSC).
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	''NOTE: Refer to Fig. 1 when setting up the TurboBlotter System.''		''NOTE: Refer to Fig. 1 when setting up the 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.
	#Place 20 sheets of dry GB004 blotting paper (thick) in stack tray.		#Place 20 sheets of dry GB004 blotting paper (thick) in stack tray.
	#Place 4 sheets of dry GB002 blotting paper (thin) on top of stack.		#Place 4 sheets of dry GB002 blotting paper (thin) on top of stack.
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	===Hybridization (2 hours + overnight + >1 hour)===		===Hybridization (2 hours + overnight + >1 hour)===
	# If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL conical 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 conical 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 conical 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 conical tube, use large glass tubes and Baker Lab hybridization oven.
-	#Prewash Blot in 0.1x SSC/ 0.1% SDS for 1 hour at ~~65�C~~ in hybridization oven. 10 - 15 mL of solution is required to cover the blot in a 50 mL tube, and sufficient for all steps. This incubation can be cut to 30 min if pressed ( says Sean.)	+	#Prewash Blot in 0.1x SSC/ 0.1% SDS for 1 hour at 65°C in hybridization oven. 10 - 15 mL of solution is required to cover the blot in a 50 mL tube, and sufficient for all steps. This incubation can be cut to 30 min if pressed ( says Sean.)
	#Remove Prewash		#Remove Prewash
-	#Prehybridize blot for > 1 hour in 10 � 15 mL of pre-hybridization solution at hybridization temp. Hybridization temp ~~?15�C~~ below estimated Tm of probe. If reusing probe in hybridization solution, thaw probe as balance. ''NOTE: It is possible to store blots in prehybridization solution sort-term to indefinitely at ~~4�C~~ or ~~�20�C~~ (Sean)''	+	#Prehybridize blot for > 1 hour in 10 - 15 mL of pre-hybridization solution at hybridization temp. Hybridization temp ~15°C below estimated Tm of probe. If reusing probe in hybridization solution, thaw probe as balance. ''NOTE: It is possible to store blots in prehybridization solution sort-term to indefinitely at 4°C or -20°C (Sean)''
	#Remove prehybridization buffer if reusing probe, otherwise retain same buffer.		#Remove prehybridization buffer if reusing probe, otherwise retain same buffer.
	#Add hybridization buffer with old probe to blot or add new probe to pre-hybridization buffer. Hybridize >6 hrs at hybridization temp.		#Add hybridization buffer with old probe to blot or add new probe to pre-hybridization buffer. Hybridize >6 hrs at hybridization temp.
-	#Pour off probe either into 32P liquid waste (remember to record waste) or into tube for storage at ~~�20�C~~ for later reuse. ''NOTE: is storing probe in 13 mL conical tube, remember not to fill tube completely.''	+	#Pour off probe either into 32P liquid waste (remember to record waste) or into tube for storage at -20°C for later reuse. ''NOTE: if storing probe in 13 mL conical tube, remember not to fill tube completely.''
-	#Wash blot in 10 � 15 mL of 6x SSC/ 0.1% SDS for 5 min at room temp in the hybridization oven (Leave door open to change temp quickly. Put shield up.)	+	#Wash blot in 10 - 15 mL of 6x SSC/ 0.1% SDS for 5 min at room temp in the hybridization oven (Leave door open to change temp quickly. Put shield up.)
	#Repeat wash twice for a total of three room temp washes. Dispose of the first wash in the 32P liquid waste. For subsequent washes pour off buffer in the sink. (Record waste.)		#Repeat wash twice for a total of three room temp washes. Dispose of the first wash in the 32P liquid waste. For subsequent washes pour off buffer in the sink. (Record waste.)
	#During washes, pre-erase PhosphorImager screen 20 min on light table.		#During washes, pre-erase PhosphorImager screen 20 min on light table.
-	#Repeat wash a forth time for 20 min at ~~10�C~~ below estimated Tm in hybridization oven.	+	#Repeat wash a forth time for 20 min at 10°C below estimated Tm in hybridization oven.
	#Pour off final wash. Remove damp blot from tube and lay on clean saran wrap. Fold saran wrap to seal blot.		#Pour off final wash. Remove damp blot from tube and lay on clean saran wrap. Fold saran wrap to seal blot.
	#Expose wrapped blot to PhosphorImager screen in casette.		#Expose wrapped blot to PhosphorImager screen in casette.
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	===Prewash Solution (0.1x SSC/ 0.1% SDS)===		===Prewash Solution (0.1x SSC/ 0.1% SDS)===
	50 mL Total Volume		50 mL Total Volume
-	#*250 ?L of 20x SSC	+	#*250 µL of 20x SSC
-	#*500 ?L of 10% SDS	+	#*500 µL of 10% SDS
	#*49.25 mL of nuclease-free H2O		#*49.25 mL of nuclease-free H2O