IGEM:Harvard/2007/Protocols - Revision history

Perry: /* General outline */

2007-08-25T00:00:28Z

General outline

←Older revision		Revision as of 00:00, 25 August 2007
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	Pour into gel frame, use comb (15- or 20-tooth) to sweep bubbles to the bottom, set comb. Let gel solidify for 10-15 minutes.		Pour into gel frame, use comb (15- or 20-tooth) to sweep bubbles to the bottom, set comb. Let gel solidify for 10-15 minutes.
-
-	~~==Post-staining==~~
-	~~If you do not have any stain already in your agarose gel, you need to perform a post-stain to visualize your DNA bands.~~
-
-	Make a mixture of TBE and staining agent, enough to submerge your gel in a shallow tray. 100ml should be enough for a 100ml agarose gel. If you used a different buffer like TAE for your gel, use the same buffer as that.
-	*5ul ethidium bromide for 100ml TBE.
-	*1:10,000 dilution of SYBR Gold, so 10ul for 100ml TBS. SYBR Gold is kept at -20dC and is light-sensitive, so make/use aliquots in tubes covered with aluminum foil.
-	~~Let the gel sit submerged in the stain, in a shallow tray, on a rocker, for 20-30min at room temperature.~~

	==Running a gel electrophoresis==		==Running a gel electrophoresis==
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	*DNA "runs to the red" so your DNA will be migrating down the gel towards the red side of the apparatus, which should thus be on the bottom end.		*DNA "runs to the red" so your DNA will be migrating down the gel towards the red side of the apparatus, which should thus be on the bottom end.

-	Pour ~~TBE~~ (~~Tris-Borate-EDTA~~) ~~buffer~~ into the apparatus.	+	Pour buffer (same as you used to make your gel) into the apparatus.
	*Don't pour directly onto the gel, as it will cause the gel to slide off. Just pour into each well. The buffer should be just covering the entire gel, until there's an even liquid surface above the wells.		*Don't pour directly onto the gel, as it will cause the gel to slide off. Just pour into each well. The buffer should be just covering the entire gel, until there's an even liquid surface above the wells.

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	*Black to black, red to red.		*Black to black, red to red.
	*You'll see bubbles at both ends. That means it's working.		*You'll see bubbles at both ends. That means it's working.
-	*You want to run it for long enough that multiple bands are well-separated, but not too long that DNA runs off the gel.	+	*You want to run it for long enough that multiple bands are well-separated, but not too long that DNA runs off the gel. The bromophenol blue in the loading dye runs with the 300bp band; this blue band should travel approximately 3/4 of the way down the gel.
		+	*You may change the voltage depending on time constraints. For example, 100V for 80min, or 150V for 30min. Anything above 150V runs risk of melting the gel.

-	After the gel has finished running, put on gloves and take out the gel in the clear plastic frame. Pour off buffer from the top back into the box or onto a napkin. Take it to the UV imager.	+	After the gel has finished running, put on gloves and take out the gel in the clear plastic frame. Pour off buffer from the top back into the box or onto a napkin. Take it to the UV imager if you have ethidium bromide stain already in the gel. If you plan to post-stain, read the post-staining section.

		+	==Post-staining==
		+	If you do not have any stain already in your agarose gel, you need to perform a post-stain to visualize your DNA bands.
		+
		+	Make a mixture of TBE and staining agent, enough to submerge your gel in a shallow tray. 100ml should be enough for a 100ml agarose gel. If you used a different buffer like TAE for your gel, use the same buffer as that.
		+	*5ul ethidium bromide for 100ml TBE.
		+	*1:10,000 dilution of SYBR Gold, so 10ul for 100ml TBS. SYBR Gold is kept at -20dC and is light-sensitive, so make/use aliquots in tubes covered with aluminum foil.
		+	Let the gel sit submerged in the stain, in a shallow tray, on a rocker, for 20-30min at room temperature.
		+
		+	If you used ethidium bromide as a stain, take your gel to the UV imager. If you used SYBR Gold as a stain, take your gel to the blue-light Safe Imager.
		+
		+	==Using the UV imager==
	Open "Quantity One" and under "File" click "Gel Doc EQ."		Open "Quantity One" and under "File" click "Gel Doc EQ."

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	Click "Save" to save the gel image with your initials, the date, what it is, in the Project 3 folder. Under "File," click "export to JPEG..." and save it as a jpg image, which you can then email to yourself or upload to your wiki page.		Click "Save" to save the gel image with your initials, the date, what it is, in the Project 3 folder. Under "File," click "export to JPEG..." and save it as a jpg image, which you can then email to yourself or upload to your wiki page.

-	If you need to cut a band, put gloves and a labcoat on. Open the drawer, install the plastic UV guard, and turn on the ~~Trans~~ UV. Look vertically down at the gel, and with a scalpel, cut into the agar as close around the band as you can without cutting into the band.	+	==Using the blue-light Safe Imager==
		+	Place your SYBR stained gel on the smooth surface of the Safe Imager. Please the square orange plastic cover over the gel, or put on the orange goggles. Turn on the blue light. Your DNA bands should be whitish-yellow against a light-green background.
		+
		+	==Gel excision==
		+	If you need to cut a band at the UV imager, put gloves and a labcoat on. Open the drawer, install the plastic UV guard, turn off the lab lights, and turn on the Prep UV. Look vertically down at the gel, and with a scalpel, cut into the agar as close around the band as you can without cutting into the band.
		+
		+	If you need to cut a band at the Safe Imager, put on the orange goggles before making your excision.

	Preweigh a 1.5ml tube, and record the mass on the tube. Use a scalpel, forceps, or pipette tip to take out the gel slice and push it into the tube.		Preweigh a 1.5ml tube, and record the mass on the tube. Use a scalpel, forceps, or pipette tip to take out the gel slice and push it into the tube.
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	==Sequencing==		==Sequencing==
-	Determine how many sequencing reactions you will be sending out, and ask Alain for a purchase order number for Genewiz, a DNA sequencing service.	+	Determine how many sequencing reactions you will be sending out, and ask Alain for a purchase order number for Genewiz, a DNA sequencing service. You can ask him for either standard service (usually within two days) or same-day service (within one day).

-	Take as many 8-tube strips as you need, and label them with ~~your initials and a three-digit number, e.g.~~ "~~PT001~~, ~~PT002..~~.". Plan and record which sequencing reactions you'll put into which tubes.	+	Take as many 8-tube strips as you need, and label them with "AV01, AV02, etc." Plan and record which sequencing reactions you'll put into which tubes.

	Into each tube, pipette 8ul of the DNA to be sequenced. If you're premixing your own primer, add 4ul primer (2uM).		Into each tube, pipette 8ul of the DNA to be sequenced. If you're premixing your own primer, add 4ul primer (2uM).
-	*Each sequencing reaction should contain only one primer. For each DNA sequence you want to be analyzed, you ~~should~~ have two sequencing reactions, one with forward primer and one with reverse primer.	+	*Each sequencing reaction should contain only one primer. For each DNA sequence you want to be analyzed, you usually will have two sequencing reactions, one with forward primer and one with reverse primer. You may need only one reaction if you only care about one section, or more than two if the section is too long.
	*You may leave out primer if you are using a vector with annealing sites for universal primers. For example, the Topo vector has M13F and M13R primer annealing sites flanking the cloning site, and Genewiz can add those primers for you.		*You may leave out primer if you are using a vector with annealing sites for universal primers. For example, the Topo vector has M13F and M13R primer annealing sites flanking the cloning site, and Genewiz can add those primers for you.

	Snap the tubes closed, wrap the strip in parafilm, and seal in a Ziplock bag.		Snap the tubes closed, wrap the strip in parafilm, and seal in a Ziplock bag.

-	Log into www.genewiz.com with Alain's email aviel@fas, and password ******.	+	Log into www.genewiz.com with Alain's email aviel@fas, and obtain the password from Alain.
-		+
-	~~Click "Place DNA Sequencing Order"~~.	+

-	~~Fill in "Number of Reactions", select "Premix", and select yes or no for "Same Day Service".~~ Click "~~Submit~~".	+	Click "Place Order".

-	~~Fill in the~~ "~~PO Number~~"~~. Fill in the sizes of your DNA samples to be sequenced. Select~~ "~~DNA Type~~", ~~usually plasmid or PCR. If you've already added primer, under Primer, select~~ "~~Already added~~", and if you ~~want Genewiz~~ to ~~add universal primers, select the proper primer~~ "~~to be added~~"~~. Review the order,~~ and click "~~Submit~~."	+	Select "Standard" or "Same day" accordingly, "Premix", and "Online form" (unless you are planning to upload an excel file). Then enter "number of reaction" and click "To Generate."
-	*If you are sequencing an insert within a plasmid, fill in the size of the entire plasmid plus insert.	+

		+	*For the corresponding samples (#1 for AV01, etc.), fill in the "DNA Name", the name of the sample you are sequencing.
		+	*Select "DNA Type", PCR product or plasmid.
		+	*Fill in the "DNA length". This should be the length of your PCR product, or the entire length of your plasmid with the insert.
		+	*If you are adding your own primer, fill it in under "My Primer". If you want Genewiz to add a universal primer, select it under "GENEWIZ primer".
		+	*Click "Next Step."
		+	*Review your order, nad click "Submit."
		+	*Fill in the "PO Number".
		+
	Print out two copies, one for Genewiz and one for yourself. Fold up the copy for Genewiz, and place into the Ziplock bag with the tubes. Drop off the bag in the dropbox at Conant 113 before 4pm Monday-Friday.		Print out two copies, one for Genewiz and one for yourself. Fold up the copy for Genewiz, and place into the Ziplock bag with the tubes. Drop off the bag in the dropbox at Conant 113 before 4pm Monday-Friday.
	*The entrance to Conant is in the walkway between Lowell Lecture Hall and Fairchild.		*The entrance to Conant is in the walkway between Lowell Lecture Hall and Fairchild.

Perry: /* Post-staining */

2007-08-24T23:38:07Z

Post-staining

←Older revision		Revision as of 23:38, 24 August 2007
Line 117:		Line 117:

	==Post-staining==		==Post-staining==
		+	If you do not have any stain already in your agarose gel, you need to perform a post-stain to visualize your DNA bands.
		+
	Make a mixture of TBE and staining agent, enough to submerge your gel in a shallow tray. 100ml should be enough for a 100ml agarose gel. If you used a different buffer like TAE for your gel, use the same buffer as that.		Make a mixture of TBE and staining agent, enough to submerge your gel in a shallow tray. 100ml should be enough for a 100ml agarose gel. If you used a different buffer like TAE for your gel, use the same buffer as that.
	*5ul ethidium bromide for 100ml TBE.		*5ul ethidium bromide for 100ml TBE.

Perry: /* Pouring a gel */

2007-08-24T23:37:31Z

Pouring a gel

←Older revision		Revision as of 23:37, 24 August 2007
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	==Pouring a gel==		==Pouring a gel==
-	For a 1% agarose gel, you need 1g agarose per ~~1mL~~ TBE buffer (Tris-Borate-EDTA). Weigh out 1g agarose powder, and pour into a gel flask, then measure 100mL ~~TBE~~ and pour into gel flask. Swirl it around some, and screw the cap on loosely.	+	For a 1% agarose gel, you need 1g agarose per 100mL 1X TBE buffer (Tris-Borate-EDTA) or TAE buffer (Tris-Acetate-EDTA). Weigh out 1g agarose powder, and pour into a gel flask, then measure 100mL buffer and pour into gel flask. Swirl it around some, and screw the cap on loosely.

	Microwave for 30-second increments, swirling in between. When it starts fizzing (usually on the 3rd time), stop the heating.		Microwave for 30-second increments, swirling in between. When it starts fizzing (usually on the 3rd time), stop the heating.

-	Cool the flask by letting it sit in cold tap water for 5-6minutes, until you can hold it comfortably in your hand. Don't let it sit too long because it will start to solidify inside the flask.	+	Cool the flask by letting it sit in cold tap water for 5-6minutes, until you can hold it comfortably in your hand. Don't let it sit too long because it will start to solidify inside the flask.
		+	*This is only necessary if you will be adding ethidium bromide to your gel, which can vaporize or inactivate if your agarose solution is too hot.

	While it's cooling, assemble gel frame: put clear plastic frame on top of creme-colored frame, between red rubber walls; turn handle to tighten. Put circle-bubble thing on top, and twist knobs on upper left and right until the bubble is in the center.		While it's cooling, assemble gel frame: put clear plastic frame on top of creme-colored frame, between red rubber walls; turn handle to tighten. Put circle-bubble thing on top, and twist knobs on upper left and right until the bubble is in the center.
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	Add 5ul ethidium bromide to the mixture, and swirl to mix. Ethidium bromide is a dangerous carcinogen because it intercalates into DNA and causes mutations.		Add 5ul ethidium bromide to the mixture, and swirl to mix. Ethidium bromide is a dangerous carcinogen because it intercalates into DNA and causes mutations.
		+	*If you are planning to post-stain your gel with ethidium bromide or SYBR Gold, do not add ethidium bromide to your gel.

	Pour into gel frame, use comb (15- or 20-tooth) to sweep bubbles to the bottom, set comb. Let gel solidify for 10-15 minutes.		Pour into gel frame, use comb (15- or 20-tooth) to sweep bubbles to the bottom, set comb. Let gel solidify for 10-15 minutes.
		+
		+	==Post-staining==
		+	Make a mixture of TBE and staining agent, enough to submerge your gel in a shallow tray. 100ml should be enough for a 100ml agarose gel. If you used a different buffer like TAE for your gel, use the same buffer as that.
		+	*5ul ethidium bromide for 100ml TBE.
		+	*1:10,000 dilution of SYBR Gold, so 10ul for 100ml TBS. SYBR Gold is kept at -20dC and is light-sensitive, so make/use aliquots in tubes covered with aluminum foil.
		+	Let the gel sit submerged in the stain, in a shallow tray, on a rocker, for 20-30min at room temperature.

	==Running a gel electrophoresis==		==Running a gel electrophoresis==

Perry at 05:58, 20 July 2007

2007-07-20T05:58:14Z

←Older revision		Revision as of 05:58, 20 July 2007
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	[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]		[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]

-	[[IGEM:Harvard/2007/Protocols/French press\|Lysing cells ~~with~~ with French press]]	+	[[IGEM:Harvard/2007/Protocols/French press\|Lysing cells with French press]]

	[[IGEM:Harvard/2007/Protocols/Protein purification\|Protein purification]]		[[IGEM:Harvard/2007/Protocols/Protein purification\|Protein purification]]

Perry at 05:58, 20 July 2007

2007-07-20T05:58:00Z

←Older revision		Revision as of 05:58, 20 July 2007
Line 22:		Line 22:

	[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]		[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]
		+
		+	[[IGEM:Harvard/2007/Protocols/French press\|Lysing cells with with French press]]
		+
		+	[[IGEM:Harvard/2007/Protocols/Protein purification\|Protein purification]]

	---------------------------------------------------------------------------------------------------		---------------------------------------------------------------------------------------------------

KMagic246 at 14:03, 13 July 2007

2007-07-13T14:03:00Z

←Older revision		Revision as of 14:03, 13 July 2007
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	[[IGEM:Harvard/2007/Protocols/Magnetic Separation\|Magnetic Separation]]		[[IGEM:Harvard/2007/Protocols/Magnetic Separation\|Magnetic Separation]]

		+	[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]

	---------------------------------------------------------------------------------------------------		---------------------------------------------------------------------------------------------------

KMagic246 at 20:51, 28 June 2007

2007-06-28T20:51:57Z

←Older revision		Revision as of 20:51, 28 June 2007
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	[[IGEM:Harvard/2007/Protocols/NanoDrop® ND-1000 Protocol\|NanoDrop® ND-1000 Protocol]]		[[IGEM:Harvard/2007/Protocols/NanoDrop® ND-1000 Protocol\|NanoDrop® ND-1000 Protocol]]
		+
		+	[[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol\|Indirect Magnetic Labeling Protocol]]
		+
		+	[[IGEM:Harvard/2007/Protocols/Magnetic Separation\|Magnetic Separation]]
		+

	---------------------------------------------------------------------------------------------------		---------------------------------------------------------------------------------------------------

KMagic246 at 18:18, 27 June 2007

2007-06-27T18:18:08Z

←Older revision		Revision as of 18:18, 27 June 2007
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	[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]		[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]
		+
		+	[[IGEM:Harvard/2007/Protocols/Plasmid Midi and Maxi Prep\|Plasmid Midi and Maxi Prep]]
		+
		+	[[IGEM:Harvard/2007/Protocols/HiSpeed Midi and Maxi Prep\|HiSpeed Midi and Maxi Prep]]

	[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]		[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]

KMagic246 at 18:16, 27 June 2007

2007-06-27T18:16:44Z

←Older revision		Revision as of 18:16, 27 June 2007
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	[[IGEM:Harvard/2007/Protocols/Transformation Protocol\|Transformation Protocol]]		[[IGEM:Harvard/2007/Protocols/Transformation Protocol\|Transformation Protocol]]

-	[[IGEM:Harvard/2007/Protocols/Plasmid ~~Prep~~ Protocol\|Plasmid ~~Prep~~ Protocol]]	+	[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]

	[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]		[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]

Perry: /* Sequencing */

2007-06-25T16:57:10Z

Sequencing

←Older revision		Revision as of 16:57, 25 June 2007
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	Snap the tubes closed, wrap the strip in parafilm, and seal in a Ziplock bag.		Snap the tubes closed, wrap the strip in parafilm, and seal in a Ziplock bag.

-	Log into www.genewiz.com with Alain's email aviel@fas, and password ~~"seqforstu" (sequencing for students)~~.	+	Log into www.genewiz.com with Alain's email aviel@fas, and password ******.

	Click "Place DNA Sequencing Order".		Click "Place DNA Sequencing Order".

←Older revision		Revision as of 05:58, 20 July 2007
Line 23:		Line 23:
	[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]		[[IGEM:Harvard/2007/Protocols/Fluorescent Labeling Protocol\|Fluorescent Labeling Protocol]]

-	[[IGEM:Harvard/2007/Protocols/French press\|Lysing cells ~~with~~ with French press]]	+	[[IGEM:Harvard/2007/Protocols/French press\|Lysing cells with French press]]

	[[IGEM:Harvard/2007/Protocols/Protein purification\|Protein purification]]		[[IGEM:Harvard/2007/Protocols/Protein purification\|Protein purification]]

←Older revision		Revision as of 18:18, 27 June 2007
Line 2:		Line 2:

	[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]		[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]
		+
		+	[[IGEM:Harvard/2007/Protocols/Plasmid Midi and Maxi Prep\|Plasmid Midi and Maxi Prep]]
		+
		+	[[IGEM:Harvard/2007/Protocols/HiSpeed Midi and Maxi Prep\|HiSpeed Midi and Maxi Prep]]

	[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]		[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]

←Older revision		Revision as of 18:16, 27 June 2007
Line 1:		Line 1:
	[[IGEM:Harvard/2007/Protocols/Transformation Protocol\|Transformation Protocol]]		[[IGEM:Harvard/2007/Protocols/Transformation Protocol\|Transformation Protocol]]

-	[[IGEM:Harvard/2007/Protocols/Plasmid ~~Prep~~ Protocol\|Plasmid ~~Prep~~ Protocol]]	+	[[IGEM:Harvard/2007/Protocols/Plasmid MiniPrep Protocol\|Plasmid MiniPrep Protocol]]

	[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]		[[IGEM:Harvard/2007/Protocols/Vector Dephosphorylation Protocol\|Vector Dephosphorylation Protocol]]