IGEM:Harvard/2010/General Protocols - Revision history

Devin R. Burrill: /* Microbiology */

Devin R. Burrill — Thu, 01 Jul 2010 18:54:37 GMT

Microbiology

←Older revision		Revision as of 18:54, 1 July 2010
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	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]
	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]
-	*[[IGEM:Harvard/2010/General Protocols/~~CompetentAgrobacterium \|~~ Agrobacterium Electroporation]]	+	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation \| Electroporating Agrobacterium]]

	=Plants=		=Plants=
	*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]		*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]
	*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]		*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]

Devin R. Burrill: /* Microbiology */

Devin R. Burrill — Thu, 01 Jul 2010 18:52:31 GMT

Microbiology

←Older revision		Revision as of 18:52, 1 July 2010
Line 19:		Line 19:
	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]
	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]
		+	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Agrobacterium Electroporation]]

	=Plants=		=Plants=
	*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]		*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]
	*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]		*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]

ChristinaAgapakis at 15:35, 15 June 2010

ChristinaAgapakis — Tue, 15 Jun 2010 15:35:04 GMT

←Older revision		Revision as of 15:35, 15 June 2010
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	*[[Silver: Bacterial Transformation \| Bacterial Transformation]]		*[[Silver: Bacterial Transformation \| Bacterial Transformation]]
	*[[Silver: Plasmid Verification \| Plasmid Verification]]		*[[Silver: Plasmid Verification \| Plasmid Verification]]
		+	*[http://labs.fhcrc.org/fero/Protocols/RNeasy_Mini_Handbook.pdf Qiagen RNeasy Plant Mini Kit]
		+	*[http://cshprotocols.cshlp.org/cgi/reprint/2007/4/pdb.kit23.pdf iScript cDNA Synthesis]

	=Microbiology=		=Microbiology=

ChristinaAgapakis at 14:59, 7 June 2010

ChristinaAgapakis — Mon, 07 Jun 2010 14:59:56 GMT

Show changes

ChristinaAgapakis: /* How Do BioBricks Work? */

ChristinaAgapakis — Mon, 07 Jun 2010 14:43:06 GMT

How Do BioBricks Work?

←Older revision		Revision as of 14:43, 7 June 2010
Line 2:		Line 2:
	A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream to the left; SpeI (S), NotI (N), PstI (P) downstream to the right.		A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream to the left; SpeI (S), NotI (N), PstI (P) downstream to the right.

-	*gaattc(E) gcggccgc(N) tctag(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)	+	*Standard Assembly of BioBricks is explained on the [http://partsregistry.org/Assembly:Standard_assembly Parts Registry Website]
		+
		+	*The sequence of the BioBrick cloning site is: gaattc(E) gcggccgc(N) tctag(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)

	*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"		*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"

Devin R. Burrill: /* How Do BioBricks Work? */

Devin R. Burrill — Thu, 03 Jun 2010 18:18:32 GMT

How Do BioBricks Work?

←Older revision		Revision as of 18:18, 3 June 2010
Line 6:		Line 6:
	*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"		*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"

-	*The BioBrick part itself must NOT contain any of the standard restriction sites, or else it will be chopped up into pieces during assembly.	+	*The BioBrick part itself must NOT contain any of the standard restriction sites, or else it will be chopped up into pieces during assembly

-	~~We commonly use two~~ Biobrick ~~vectors: V0120 (same as V0100) and V0002. The V0120~~ vector ~~preferred over the V0002 vector because~~ V0120 is a high-copy plasmid (~300 ng/uL from a mini-prep of a 5 mL culture) ~~while V0002 is not. The V0002 plasmid is most frequently found in older parts.~~	+	*The Biobrick vector we will be using is called V0120, a high-copy plasmid (~300 ng/uL from a mini-prep of a 5 mL culture)
-	~~The vector backbone of V0100 & V0120 are identical. The only difference is that the V0120 vector contains a part in between the Biobrick ends, while the V0100 does not.~~	+
-	The V0120 vector contains a part composed of the coding regions for the ccdB death gene and chloramphenicol resistance. Because of this, the V0120 vector can be digested, pcr purified and used directly for ligations. Treating with CIP or gel purifying the vector is unnecessary, because transformation of any intact V0120 plasmid into normal TOP10 competent cells (purple lid) will kill the host. V0120 can be successfully propagated in TOP10 Survival competent cells (green lid).	+
-	~~V0120 is part# 116 in our internal database. Its annotated sequence can be found here.~~	+

	=Growing a Bacterial E. coli Culture=		=Growing a Bacterial E. coli Culture=

Devin R. Burrill: /* How Do BioBricks Work? */

Devin R. Burrill — Thu, 03 Jun 2010 17:28:31 GMT

How Do BioBricks Work?

←Older revision		Revision as of 17:28, 3 June 2010
Line 5:		Line 5:

	*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"		*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"
		+
		+	*The BioBrick part itself must NOT contain any of the standard restriction sites, or else it will be chopped up into pieces during assembly.
		+
		+	We commonly use two Biobrick vectors: V0120 (same as V0100) and V0002. The V0120 vector preferred over the V0002 vector because V0120 is a high-copy plasmid (~300 ng/uL from a mini-prep of a 5 mL culture) while V0002 is not. The V0002 plasmid is most frequently found in older parts.
		+	The vector backbone of V0100 & V0120 are identical. The only difference is that the V0120 vector contains a part in between the Biobrick ends, while the V0100 does not.
		+	The V0120 vector contains a part composed of the coding regions for the ccdB death gene and chloramphenicol resistance. Because of this, the V0120 vector can be digested, pcr purified and used directly for ligations. Treating with CIP or gel purifying the vector is unnecessary, because transformation of any intact V0120 plasmid into normal TOP10 competent cells (purple lid) will kill the host. V0120 can be successfully propagated in TOP10 Survival competent cells (green lid).
		+	V0120 is part# 116 in our internal database. Its annotated sequence can be found here.

	=Growing a Bacterial E. coli Culture=		=Growing a Bacterial E. coli Culture=

Devin R. Burrill: /* How Do BioBricks Work? */

Devin R. Burrill — Thu, 03 Jun 2010 17:22:01 GMT

How Do BioBricks Work?

←Older revision		Revision as of 17:22, 3 June 2010
Line 1:		Line 1:
	=How Do BioBricks Work?=		=How Do BioBricks Work?=
-	A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream left; SpeI (S), NotI (N), PstI (P) downstream right.	+	A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream to the left; SpeI (S), NotI (N), PstI (P) downstream to the right.

-	*gaattc(E) gcggccgc(N) ~~atctag~~(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)	+	*gaattc(E) gcggccgc(N) tctag(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)
		+
		+	*E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"

	=Growing a Bacterial E. coli Culture=		=Growing a Bacterial E. coli Culture=

Devin R. Burrill: /* How Do BioBricks Work? */

Devin R. Burrill — Thu, 03 Jun 2010 17:19:35 GMT

How Do BioBricks Work?

←Older revision		Revision as of 17:19, 3 June 2010
Line 2:		Line 2:
	A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream left; SpeI (S), NotI (N), PstI (P) downstream right.		A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream left; SpeI (S), NotI (N), PstI (P) downstream right.

-	*gaattc gcggccgc atctag [BioBrick Part] actagt gcggccg ctgcag	+	*gaattc(E) gcggccgc(N) atctag(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)
-	*EcoRI NotI XbaI SpeI NotI PstI	+

	=Growing a Bacterial E. coli Culture=		=Growing a Bacterial E. coli Culture=

Devin R. Burrill: /* How Do BioBricks Work? */

Devin R. Burrill — Thu, 03 Jun 2010 17:18:43 GMT

How Do BioBricks Work?

←Older revision		Revision as of 17:18, 3 June 2010
Line 2:		Line 2:
	A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream left; SpeI (S), NotI (N), PstI (P) downstream right.		A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream left; SpeI (S), NotI (N), PstI (P) downstream right.

-	gaattc gcggccgc atctag [BioBrick Part] actagt gcggccg ctgcag	+	*gaattc gcggccgc atctag [BioBrick Part] actagt gcggccg ctgcag
-	EcoRI NotI XbaI SpeI NotI PstI	+	*EcoRI NotI XbaI SpeI NotI PstI

	=Growing a Bacterial E. coli Culture=		=Growing a Bacterial E. coli Culture=

←Older revision		Revision as of 18:54, 1 July 2010
Line 19:		Line 19:
	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]
	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]
-	*[[IGEM:Harvard/2010/General Protocols/~~CompetentAgrobacterium \|~~ Agrobacterium Electroporation]]	+	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation \| Electroporating Agrobacterium]]

	=Plants=		=Plants=
	*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]		*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]
	*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]		*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]

←Older revision		Revision as of 18:52, 1 July 2010
Line 19:		Line 19:
	*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/Agrobacterium \| Growing Agrobacterium]]
	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]		*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Making Electro-Competent Agrobacterium]]
		+	*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium \| Agrobacterium Electroporation]]

	=Plants=		=Plants=
	*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]		*[[IGEM:Harvard/2010/General Protocols/Arabidopsis \| Growing Arabidopsis]]
	*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]		*[[IGEM:Harvard/2010/General Protocols/FlowerDipping \| Arabidopsis transformation via Flower Dipping]]