Polyacrylamide gel electrophoresis - Revision history

Michael A. Speer at 19:02, 29 March 2011

2011-03-29T19:02:26Z

←Older revision		Revision as of 19:02, 29 March 2011
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-
-	~~==Acknowledgments==~~
-	~~Acnkowledge any help you had in development, testing, writing this protocol.~~

	==References==		==References==

Michael A. Speer: /* References */

2011-03-29T19:01:46Z

References

←Older revision		Revision as of 19:01, 29 March 2011
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	==References==		==References==
-	~~See [[OpenWetWare~~:~~Biblio]] for information on how to reference within~~ a ~~wiki~~.	+	*Sambrook J, Russell DW, Cold Spring Harbor L: Molecular cloning : a laboratory manual / Joseph Sambrook, David W. Russell. Cold Spring Harbor, N.Y. :: Cold Spring Harbor Laboratory; 2001.
-		+

	==Discussion==		==Discussion==

Michael A. Speer: /* Procedure */

2011-03-29T18:57:51Z

Procedure

Michael A. Speer: /* Notes */

2011-03-29T18:53:03Z

Notes

←Older revision		Revision as of 18:53, 29 March 2011
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	*For thicker gels (bigger wells) more gel will need to be made. The amount of gel necessary can be calculated volumetrically.		*For thicker gels (bigger wells) more gel will need to be made. The amount of gel necessary can be calculated volumetrically.
	*The voltage applied to the gel will vary according the the tube of gel you cast. The specified voltage is 1-8 volts/cm. Centimeters in this case specifies the length of the gel from top to bottom (i.e the direction the DNA will travel).		*The voltage applied to the gel will vary according the the tube of gel you cast. The specified voltage is 1-8 volts/cm. Centimeters in this case specifies the length of the gel from top to bottom (i.e the direction the DNA will travel).
-	To determine the final acrylamide concentration for your application use the following table. For example: if I had a 40% acrylamide stock solution and I wanted 5mL of gel with a final concentration of 12% acrylamide, then I would add 5(12/40)=1.5mL of acrylamide solution to make my gel.	+	To determine the final acrylamide concentration for your application use the following table. For example: if I had a 40% acrylamide stock solution and I wanted 5mL of gel with a final concentration of 12% acrylamide, then I would add 5ml(12%/40%)=1.5mL of acrylamide solution to make my gel.

	<center>		<center>

Reuben Cutfield: Polyacrylamide Gel Electrophoresis moved to Polyacrylamide gel electrophoresis: Following OWW naming conventions.

2010-12-23T21:57:49Z

Polyacrylamide Gel Electrophoresis moved to Polyacrylamide gel electrophoresis: Following OWW naming conventions.

←Older revision

Revision as of 21:57, 23 December 2010

Michael A. Speer at 21:20, 17 June 2009

2009-06-17T21:20:55Z

←Older revision		Revision as of 21:20, 17 June 2009
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	See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.		See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.

-	~~==Specific Protocols==~~
-	~~Add links to all the OWW protocols that have been used in making the consensus.~~

	==Discussion==		==Discussion==
	You can [[Talk:{{PAGENAME}}\|discuss this protocol]].		You can [[Talk:{{PAGENAME}}\|discuss this protocol]].

-	~~Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.~~

	[[Category:Protocol]]		[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Michael A. Speer: /* Notes */

2009-06-17T21:19:24Z

Notes

←Older revision		Revision as of 21:19, 17 June 2009
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	! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)		! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)
	\|-		\|-
-	\| align="center"\|3.5 \|\| 1000-2000	+	\| align="center"\|3.5 \|\| align="center"\|1000-2000
	\|-		\|-
-	\| 5.0 \|\| 80-500	+	\| align="center"\|5.0 \|\| align="center"\|80-500
	\|-		\|-
-	\| 8.0\|\| 60-400	+	\| align="center"\|8.0\|\| align="center"\|60-400
	\|-		\|-
-	\| 12.0 \|\| 40-200	+	\| align="center"\|12.0 \|\| align="center"\|40-200
	\|-		\|-
-	\| 15.0 \|\| 25-150	+	\| align="center"\|15.0 \|\| align="center"\|25-150
	\|-		\|-
-	\| 20.0 \|\| 6-100	+	\| align="center"\|20.0 \|\| align="center"\|6-100
	\|}		\|}
	</center>		</center>

Michael A. Speer: /* Notes */

2009-06-17T21:18:14Z

Notes

←Older revision		Revision as of 21:18, 17 June 2009
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	! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)		! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)
	\|-		\|-
-	\| 3.5 \|\| 1000-2000	+	\| align="center"\|3.5 \|\| 1000-2000
	\|-		\|-
	\| 5.0 \|\| 80-500		\| 5.0 \|\| 80-500

Michael A. Speer: /* Notes */

2009-06-17T21:16:37Z

Notes

←Older revision		Revision as of 21:16, 17 June 2009
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	<center>		<center>
-	{\| border="1"	+	{\| border="1" cellpadding="5" cellspacing="0" align="center"
	\|+ '''Choosing an Acrylamide Concentration'''		\|+ '''Choosing an Acrylamide Concentration'''
	! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)		! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)

Michael A. Speer: /* Notes */

2009-06-17T21:15:42Z

Notes

←Older revision		Revision as of 21:15, 17 June 2009
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	<center>		<center>
	{\| border="1"		{\| border="1"
-	\|+	+	\|+ '''Choosing an Acrylamide Concentration'''
	! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)		! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)
	\|-		\|-
-	\| ~~align="center"~~ 3.5 \|\| 1000-2000	+	\| 3.5 \|\| 1000-2000
	\|-		\|-
	\| 5.0 \|\| 80-500		\| 5.0 \|\| 80-500

←Older revision		Revision as of 18:57, 29 March 2011
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	:*Y mL water (To make 5 mL)<br>		:*Y mL water (To make 5 mL)<br>
	:*2µl TEMED<br>		:*2µl TEMED<br>
-	3. Pipet the acrylamide solution between the casting plates using a 5ml pipetor<br>	+	3. Pipet the acrylamide solution between the casting plates using a 5ml pipetor.<br>
-	4. Insert comb into the top of the gel and allow it to cure vertically for approximately 30 minutes<br>	+	4. Insert comb into the top of the gel and allow it to cure vertically for approximately 30 minutes.<br>
-	5. Combine the following for all DNA samples including the ladder<br>	+	5. Combine the following for all DNA samples including the ladder:<br>
	:*10μL DNA solution<br>		:*10μL DNA solution<br>
	:*1μL SYBR green (100X Dilution in DMSO)<br>		:*1μL SYBR green (100X Dilution in DMSO)<br>
	:*1μL Bromophenol Blue<br>		:*1μL Bromophenol Blue<br>
-	6. Insert the gel into the electrophoresis chamber allong with the buffer dam	+	6. Insert the gel into the electrophoresis chamber allong with the buffer dam.
-	:*Make sure both the gel and the buffer dam seal	+	:*Make sure both the gel and the buffer dam seal.
-	:*The wells on the gel should face the inside	+	:*The wells on the gel should face the inside.
-	7. Add 1X TBE to the space between the gel and tue buffer dam until the TBE fills the wells in the gel<br>	+	7. Add 1X TBE to the space between the gel and tue buffer dam until the TBE fills the wells in the gel.<br>
-	:*No TBE should leak into the space outside of this chamber	+	:*No TBE should leak into the space outside of this chamber.
-	8. Add 1X TBE to the outer chamber to the specified fill level.	+	8. Add 1X TBE to the outer chamber to the specified fill level.<br>
-	8. Add DNA mix to wells. <br>	+	9. Add DNA mix to wells. <br>
-	9. Apply 80 volts and run for approximately 60 minutes<br>	+	10. Apply 80 volts and run for approximately 60 minutes.<br>
-		+
-		+

	==Notes==		==Notes==