IGEM:Harvard/2006/Lab Intro - Revision history

ShawnDouglas: /* June 12 */

2006-06-14T12:42:55Z

June 12

←Older revision		Revision as of 12:42, 14 June 2006
Line 114:		Line 114:
	*17:00 - image gels, plan for tomorrow		*17:00 - image gels, plan for tomorrow

-	[[Folding DNA nanostructures]]	+	[[IGEM:Harvard/2006/Folding_DNA_nanostructures\|Folding DNA nanostructures]]

	==June 13==		==June 13==

2006-06-12T17:12:28Z

June 12

←Older revision		Revision as of 17:12, 12 June 2006
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	*17:00 - image gels, plan for tomorrow		*17:00 - image gels, plan for tomorrow

-	~~'''~~Folding DNA ~~nanotubes'''~~	+	[[Folding DNA nanostructures]]
-	*working stocks	+
-	**44 nM scaffold (20 {{ul}})	+
-	**0.99 {{um}} each oligo	+
-	*goal	+
-	**10 nM scaffold	+
-	**100 nM each oligo	+
-	**20 {{ul}} reaction volume	+
-	*calculations	+
-	*scaffold = (10 nM)/(44 nM) 20 {{ul}} = 4.5 {{ul}}	+
-	*oligos = (100 nM)/(990 nM) 20 {{ul}} = 2 {{ul}}	+
-	*reaction mixture	+
-	**4.5 {{ul}} p7308 scaffold (44 nM)	+
-	**2 {{ul}} oligos (990 nM)	+
-	**2 {{ul}} 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM {{mgcl2}})	+
-	**11.5 {{ul}} d{{h2o}}	+
-	*annealing protocol	+
-	**90{{c}} 5' → 65{{c}} 20' → 55{{c}} 20' → 45{{c}} 20' → 37{{c}} 30'	+

	==June 13==		==June 13==

2006-06-12T17:09:06Z

June 12

←Older revision		Revision as of 17:09, 12 June 2006
Line 113:		Line 113:
	*16:00 - load and run gels for folding reactions		*16:00 - load and run gels for folding reactions
	*17:00 - image gels, plan for tomorrow		*17:00 - image gels, plan for tomorrow
		+
		+	'''Folding DNA nanotubes'''
		+	*working stocks
		+	**44 nM scaffold (20 {{ul}})
		+	**0.99 {{um}} each oligo
		+	*goal
		+	**10 nM scaffold
		+	**100 nM each oligo
		+	**20 {{ul}} reaction volume
		+	*calculations
		+	*scaffold = (10 nM)/(44 nM) 20 {{ul}} = 4.5 {{ul}}
		+	*oligos = (100 nM)/(990 nM) 20 {{ul}} = 2 {{ul}}
		+	*reaction mixture
		+	**4.5 {{ul}} p7308 scaffold (44 nM)
		+	**2 {{ul}} oligos (990 nM)
		+	**2 {{ul}} 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM {{mgcl2}})
		+	**11.5 {{ul}} d{{h2o}}
		+	*annealing protocol
		+	**90{{c}} 5' → 65{{c}} 20' → 55{{c}} 20' → 45{{c}} 20' → 37{{c}} 30'

	==June 13==		==June 13==

2006-06-09T19:29:49Z

June 9

←Older revision		Revision as of 19:29, 9 June 2006
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	*Qiagen gel-extraction kit. Quantitate on nanodrop to determine concentration. (see http://openwetware.org/wiki/Wittrup:_Gel_extraction and Qiagen manual).		*Qiagen gel-extraction kit. Quantitate on nanodrop to determine concentration. (see http://openwetware.org/wiki/Wittrup:_Gel_extraction and Qiagen manual).
	*(pour a gel at this point)		*(pour a gel at this point)
-	*Ligation (follow protocol that comes with T4 ligase, for example), with proper controls. While ligation runs, figure out what size products you are expecting.	+	*[[Knight:DNA_ligation_using_NEB_Quick_Ligation_Kit\|Ligation]] (follow protocol that comes with T4 ligase, for example), with proper controls. While ligation runs, figure out what size products you are expecting.
	*Gel purification of ligated product. Same as steps 4-7.		*Gel purification of ligated product. Same as steps 4-7.
	*Transformation (same as Tuesday).		*Transformation (same as Tuesday).

2006-06-09T19:29:07Z

Standard Assembly Information

←Older revision		Revision as of 19:29, 9 June 2006
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	*Digest E0241 - XbaI + PstI (Suffix)		*Digest E0241 - XbaI + PstI (Suffix)

-	==Standard Assembly Information==	+	'''Standard Assembly Information'''
	[http://parts.mit.edu/wiki/index.php/Standard_Assembly Assembly Overview]		[http://parts.mit.edu/wiki/index.php/Standard_Assembly Assembly Overview]

2006-06-09T19:26:36Z

June 9

←Older revision		Revision as of 19:26, 9 June 2006
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	**Digest - X + P (part 2: suffix)		**Digest - X + P (part 2: suffix)
	**plasmid: pSB1A2 (amp resistance)		**plasmid: pSB1A2 (amp resistance)
		+
		+
		+	*[[Knight:Restriction_Digest\|BioBrick Digests]]

2006-06-09T19:08:26Z

June 9

←Older revision		Revision as of 19:08, 9 June 2006
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	==June 9==		==June 9==
	*[http://parts.mit.edu/r/parts/htdocs/Assembly/index.cgi BioBrick Assembly]		*[http://parts.mit.edu/r/parts/htdocs/Assembly/index.cgi BioBrick Assembly]
-	*[http://parts.mit.edu/r/parts/htdocs/Assembly/standard_assembly.cgi Standard Assembly of 2 ~~Part~~]	+	*[http://parts.mit.edu/r/parts/htdocs/Assembly/standard_assembly.cgi Standard Assembly of 2 Parts]

	'''Old pages'''		'''Old pages'''

2006-06-09T18:20:18Z

June 5

←Older revision		Revision as of 18:20, 9 June 2006
Line 20:		Line 20:
	*Digest E0241 - XbaI + PstI (Suffix)		*Digest E0241 - XbaI + PstI (Suffix)

-	~~Restriction Enzymes~~	+	==Standard Assembly Information==
-	*[http://~~rebase~~.~~neb~~.~~com~~/~~rebase~~/~~enz/XbaI~~.~~html About XbaI~~]	+	[http://parts.mit.edu/wiki/index.php/Standard_Assembly Assembly Overview]

-	~~[http:~~/~~/www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=592365&dopt=Abstract XbaI source]~~	+	<b>Restriction Enzymes Used</b>

-	*[http://rebase.neb.com/rebase/enz/~~SpeI~~.html About ~~SpeI~~]	+	*[http://rebase.neb.com/rebase/enz/XbaI.html About XbaI] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=592365&dopt=Abstract Origins of XbaI]

-	[http://home1.gte.net/vsjslsk1/sphaerotilus.htm SpeI ~~Source~~]	+	*[http://rebase.neb.com/rebase/enz/SpeI.html About SpeI] [http://home1.gte.net/vsjslsk1/sphaerotilus.htm Origins of SpeI]
		+
		+	*[http://rebase.neb.com/rebase/enz/EcoRI.html EcoRI] [http://en.wikipedia.org/wiki/EcoRI More info about EcoRI]
		+
		+	*[http://rebase.neb.com/rebase/enz/PstI.html PstI] [http://www.thelabrat.com/restriction/sources/Providenciastuartii.shtml Origins of Pst1]

	Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing		Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing

2006-06-09T18:12:15Z

June 5

←Older revision		Revision as of 18:12, 9 June 2006
Line 19:		Line 19:
	*Digest R0010 - SpeI + PstI (prefix)		*Digest R0010 - SpeI + PstI (prefix)
	*Digest E0241 - XbaI + PstI (Suffix)		*Digest E0241 - XbaI + PstI (Suffix)
		+
		+	Restriction Enzymes
		+	*[http://rebase.neb.com/rebase/enz/XbaI.html About XbaI]
		+
		+	[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=592365&dopt=Abstract XbaI source]
		+
		+	*[http://rebase.neb.com/rebase/enz/SpeI.html About SpeI]
		+
		+	[http://home1.gte.net/vsjslsk1/sphaerotilus.htm SpeI Source]

	Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing		Gel extraction -> Ligation -> Transformation -> Miniprep -> Sequencing

2006-06-09T17:50:55Z

June 5

←Older revision		Revision as of 17:50, 9 June 2006
Line 2:		Line 2:
	Parts:		Parts:

-	R0010 - LacI promoter	+	[http://parts.mit.edu/registry/index.php/Part:BBa_R0010 R0010] - LacI promoter
	* Plate 1, Well 7K		* Plate 1, Well 7K
	* amp resistance		* amp resistance
	* Hydrated yellow		* Hydrated yellow

-	E0241 - GFP reporter	+	[http://parts.mit.edu/registry/index.php/Part:BBa_E0241 E0241] - GFP reporter
	* Plate 2, Well 15L		* Plate 2, Well 15L
	* amp resistance		* amp resistance
	* Hydrated red		* Hydrated red

-	E7104 - GFP reporter behind T7 promoter	+	[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 E7104] E7104 - GFP reporter behind T7 promoter
	* Plate 2, Well 13F		* Plate 2, Well 13F
	* amp resistance		* amp resistance