Knight:Agarose gel electrophoresis - Revision history

Reshma P. Shetty at 20:16, 6 December 2006

2006-12-06T20:16:16Z

←Older revision		Revision as of 20:16, 6 December 2006
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	*We have replaced [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.		*We have replaced [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:DNA]]

Reshma P. Shetty: /* Interpreting results */

2006-12-06T20:15:11Z

Interpreting results

←Older revision		Revision as of 20:15, 6 December 2006
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	==Interpreting results==		==Interpreting results==

-	*If you are getting unexpected bands on your gel you may want to look at the [[~~Common~~ Agarose ~~Gel Issues~~]]	+	*If you are getting unexpected bands on your gel you may want to look at the [[Agarose gel electrophoresis/Common issues\|common agarose gel issues]].

	==Notes==		==Notes==

	*We have replaced [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.		*We have replaced [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.

Reshma P. Shetty: /* Notes */

2006-12-06T20:14:40Z

Notes

←Older revision		Revision as of 20:14, 6 December 2006
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	==Notes==		==Notes==

-	*We~~'re trying out replacing~~ [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.	+	*We have replaced [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.

Reshma P. Shetty: /* Procedure */

2006-12-06T20:14:10Z

Procedure

←Older revision		Revision as of 20:14, 6 December 2006
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	#Remove gel from gel box shaking gently to allow residual buffer to fall back into gel box.		#Remove gel from gel box shaking gently to allow residual buffer to fall back into gel box.
	#Place in middle of UV box inside gel imager (you can leave the gel in gel tray).		#Place in middle of UV box inside gel imager (you can leave the gel in gel tray).
		+	#Make sure that filter wheel is set to position 4 (SYBR gold).
	#Close the door and turn on reflective white light button.		#Close the door and turn on reflective white light button.
	#In gel imager software, click the "Acquire" button such that gel displays on screen.		#In gel imager software, click the "Acquire" button such that gel displays on screen.
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	#Save a copy of gel picture in your user folder.		#Save a copy of gel picture in your user folder.
	#Print.		#Print.
-	#Remove gel and ~~dispose~~ in ~~ethidium bromide waste container~~.	+	#Remove gel and throw in trash.
	#Wipe down UV box if necessary.		#Wipe down UV box if necessary.

Reshma P. Shetty: /* Running agarose gels */

2006-12-06T20:13:15Z

Running agarose gels

←Older revision		Revision as of 20:13, 6 December 2006
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	*Precast gel with the appropriate percentage and well size/numbers for your samples (see above)		*Precast gel with the appropriate percentage and well size/numbers for your samples (see above)
	*[[1X TAE]]		*[[1X TAE]]
-	*[[Knight:Loading dye\|Loading dye]]	+	*[[Knight:Loading dye\|Loading dye]]
-	*[[Ethidium Bromide]] (10 mg/mL stock)	+

	===Procedure===		===Procedure===
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	#Add 1X TAE buffer to gel box such that buffer just covers the top of the gel.		#Add 1X TAE buffer to gel box such that buffer just covers the top of the gel.
	#Remove comb.		#Remove comb.
-	~~#Add 10 μL ethidium bromide stock solution to the running buffer well near the positive terminal.~~
	#Load 12 μL prepared ladder <br> ''Typically load ladder in left-most lane and sometimes right-most lane as well depending on whether you have the space.''		#Load 12 μL prepared ladder <br> ''Typically load ladder in left-most lane and sometimes right-most lane as well depending on whether you have the space.''
	#Use 2 μL loading dye per 10 μL of sample.		#Use 2 μL loading dye per 10 μL of sample.

Reshma P. Shetty: /* Casting agarose gels */

2006-12-06T20:12:39Z

Casting agarose gels

←Older revision		Revision as of 20:12, 6 December 2006
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	*[[1X TAE]]		*[[1X TAE]]
-	*~~[[Ethidium Bromide]]~~ (10 ~~mg/mL~~ stock)	+	*SYBR safe (10,000X stock)
	*Agarose		*Agarose
	*Microwave		*Microwave

Reshma P. Shetty: /* Procedure */

2006-12-06T20:11:59Z

Procedure

←Older revision		Revision as of 20:11, 6 December 2006
Line 20:		Line 20:
	#Remove from microwave and let cool by either sitting on bench top or adding stir bar and placing on stir plate. <br> ''The advantage of the stir plate is that, if you forget about your gel for a while, it is less likely to solidify accidentally. <br> If you are in a hurry, you can place the bottle in a beaker of room temperature water on the stir plate to speed the cooling process significantly.''		#Remove from microwave and let cool by either sitting on bench top or adding stir bar and placing on stir plate. <br> ''The advantage of the stir plate is that, if you forget about your gel for a while, it is less likely to solidify accidentally. <br> If you are in a hurry, you can place the bottle in a beaker of room temperature water on the stir plate to speed the cooling process significantly.''
	#While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.		#While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.
-	#Once gel is cooled so that it can be touched comfortably with your gloved hand, add 15 μL ~~Ethidium Bromide~~ (~~final concentration of 0.5 μg/mL~~).	+	#Once gel is cooled so that it can be touched comfortably with your gloved hand, add 30 μL SYBR Safe (10,000X concentrate).
	#Pour gel into casting trays. <br> ''The height of the gel will depend on how much you wish to load. Diagnostic gels can be reasonably shallow since typically 10 μL volumes are loaded. For gel purifications, the gel should be deeper to enable loading of large sample volumes.''		#Pour gel into casting trays. <br> ''The height of the gel will depend on how much you wish to load. Diagnostic gels can be reasonably shallow since typically 10 μL volumes are loaded. For gel purifications, the gel should be deeper to enable loading of large sample volumes.''
	#Let gels sit until they are solidified. <br> ''Gels are solid when they are cloudy in appearance and firm to the touch.''		#Let gels sit until they are solidified. <br> ''Gels are solid when they are cloudy in appearance and firm to the touch.''

Reshma P. Shetty: /* Materials */

2006-08-25T17:18:16Z

Materials

←Older revision		Revision as of 17:18, 25 August 2006
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	*Precast gel with the appropriate percentage and well size/numbers for your samples (see above)		*Precast gel with the appropriate percentage and well size/numbers for your samples (see above)
	*[[1X TAE]]		*[[1X TAE]]
-	*[[Loading dye]]	+	*[[Knight:Loading dye\|Loading dye]]
	*[[Ethidium Bromide]] (10 mg/mL stock)		*[[Ethidium Bromide]] (10 mg/mL stock)

Reshma P. Shetty: Knight:Agarose gel moved to Knight:Agarose gel electrophoresis

2006-01-04T01:21:20Z

Knight:Agarose gel moved to Knight:Agarose gel electrophoresis

←Older revision

Revision as of 01:21, 4 January 2006

Reshma P. Shetty: /* Notes */

2005-09-21T15:13:18Z

Notes

←Older revision		Revision as of 15:13, 21 September 2005
Line 79:		Line 79:
	==Notes==		==Notes==

-	*We're trying out replacing [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain].	+	*We're trying out replacing [[Ethidium Bromide]] with [http://probes.invitrogen.com/products/sybrsafe/ SYBR Safe DNA gel stain]. According to Reet Mand at Alpha Innotech Corp Technical Support: For SybrSafe dye, we recommend Fluorescein filter which is centered at 535nm. The part number is FLSC-500 and you can order it from us. Tom says this is the same as the SYBR-Gold filter installed in position 4 of the filter wheel.