Cloning - Revision history

Christopher C Vanlang: /* Transformation */

2012-01-13T01:03:17Z

Transformation

←Older revision		Revision as of 01:03, 13 January 2012
Line 47:		Line 47:
	Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].		Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].

-	~~====~~Reagents~~====~~	+	'''Reagents'''
	*[[Competent cells]]: XL2-Blue (Stratagene 200150).		*[[Competent cells]]: XL2-Blue (Stratagene 200150).
	**Learn how to make your own by [[Preparing_chemically_competent_cells]]		**Learn how to make your own by [[Preparing_chemically_competent_cells]]
Line 53:		Line 53:
	*ß-mercaptoethanol (ß-MAE)		*ß-mercaptoethanol (ß-MAE)

-	~~====~~Method~~====~~	+	'''Method'''

	===Screening===		===Screening===

Christopher C Vanlang: /* Ligation */

2012-01-13T01:02:56Z

Ligation

←Older revision		Revision as of 01:02, 13 January 2012
Line 32:		Line 32:
	The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].		The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].

-	~~====~~Reagents~~====~~	+	'''Reagents'''
	*[[T4 DNA ligase]]		*[[T4 DNA ligase]]
	*10x T4 DNA Ligase Buffer		*10x T4 DNA Ligase Buffer
Line 38:		Line 38:
	*Purified, linearized insert (likely in H2O or EB)		*Purified, linearized insert (likely in H2O or EB)

-	~~====~~Method~~====~~	+	'''Method'''
	#Mix reagents for a 10 μL reaction.		#Mix reagents for a 10 μL reaction.
	#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C		#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C

Christopher C Vanlang: /* Materials */

2012-01-13T01:02:25Z

Materials

←Older revision		Revision as of 01:02, 13 January 2012
Line 9:		Line 9:
	*aluminium rack for PCR tubes		*aluminium rack for PCR tubes
	=== Media and plates ===		=== Media and plates ===
-	~~1.2-~~ Reagents	+	'''Reagents'''
	*[[LB]] (Luria-Bertami) agar plates		*[[LB]] (Luria-Bertami) agar plates
	*[[IPTG]] stock solution (100 nM)		*[[IPTG]] stock solution (100 nM)

Christopher C Vanlang at 01:01, 13 January 2012

2012-01-13T01:01:02Z

←Older revision		Revision as of 01:01, 13 January 2012
Line 1:		Line 1:
	{{back to protocols}}		{{back to protocols}}
-	=General Procedure=	+	==General Procedure==
	This should be a [[Help:Consensus protocol\|consensus protocol]]. See the bottom of this article for specific protocols.		This should be a [[Help:Consensus protocol\|consensus protocol]]. See the bottom of this article for specific protocols.

-	=Materials=	+	==Materials==
-	==Equipment==	+	===Equipment===
	*gas burner		*gas burner
	*rotating plate		*rotating plate
	*aluminium rack for PCR tubes		*aluminium rack for PCR tubes
-	== Media and plates ==	+	=== Media and plates ===
	1.2- Reagents		1.2- Reagents
	*[[LB]] (Luria-Bertami) agar plates		*[[LB]] (Luria-Bertami) agar plates
Line 22:		Line 22:
	**DW		**DW

-	=Procedure=	+	==Procedure==
-	==Enriching the insert==	+	===Enriching the insert===
	The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]		The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]

-	==Restriction Digest==	+	===Restriction Digest===
	Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].		Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].

-	==Ligation==	+	===Ligation===
	The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].		The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].

-	===Reagents===	+	====Reagents====
	*[[T4 DNA ligase]]		*[[T4 DNA ligase]]
	*10x T4 DNA Ligase Buffer		*10x T4 DNA Ligase Buffer
Line 38:		Line 38:
	*Purified, linearized insert (likely in H2O or EB)		*Purified, linearized insert (likely in H2O or EB)

-	===Method===	+	====Method====
	#Mix reagents for a 10 μL reaction.		#Mix reagents for a 10 μL reaction.
	#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C		#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
Line 44:		Line 44:
	#Store at -20°C.		#Store at -20°C.

-	==Transformation==	+	===Transformation===
	Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].		Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].

-	===Reagents===	+	====Reagents====
	*[[Competent cells]]: XL2-Blue (Stratagene 200150).		*[[Competent cells]]: XL2-Blue (Stratagene 200150).
	**Learn how to make your own by [[Preparing_chemically_competent_cells]]		**Learn how to make your own by [[Preparing_chemically_competent_cells]]
Line 53:		Line 53:
	*ß-mercaptoethanol (ß-MAE)		*ß-mercaptoethanol (ß-MAE)

-	===Method===	+	====Method====

-	==Screening==	+	===Screening===
	Either a [[Restriction_digest\| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]]		Either a [[Restriction_digest\| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]]

-	=Critical steps=	+	==Critical steps==

-	=Troubleshooting=	+	==Troubleshooting==
-	=Notes=	+	==Notes==
-	=See Also=	+	==See Also==
	===Cloning Software===		===Cloning Software===
	{{#dpl:category=cloning\|category=software}}		{{#dpl:category=cloning\|category=software}}

Christopher C Vanlang at 00:59, 13 January 2012

2012-01-13T00:59:45Z

←Older revision		Revision as of 00:59, 13 January 2012
Line 1:		Line 1:
	{{back to protocols}}		{{back to protocols}}
-	==General Procedure==	+	=General Procedure=
	This should be a [[Help:Consensus protocol\|consensus protocol]]. See the bottom of this article for specific protocols.		This should be a [[Help:Consensus protocol\|consensus protocol]]. See the bottom of this article for specific protocols.

-	==Materials==	+	=Materials=
-	===Equipment===	+	==Equipment==
	*gas burner		*gas burner
	*rotating plate		*rotating plate
	*aluminium rack for PCR tubes		*aluminium rack for PCR tubes
-	=== Media and plates ===	+	== Media and plates ==
	1.2- Reagents		1.2- Reagents
	*[[LB]] (Luria-Bertami) agar plates		*[[LB]] (Luria-Bertami) agar plates
Line 22:		Line 22:
	**DW		**DW

-	==Procedure==	+	=Procedure=
-	===Enriching the insert===	+	==Enriching the insert==
	The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]		The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]

-	===Restriction Digest===	+	==Restriction Digest==
	Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].		Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].

-	===Ligation===	+	==Ligation==
	The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].		The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].

-	====Reagents====	+	===Reagents===
	*[[T4 DNA ligase]]		*[[T4 DNA ligase]]
	*10x T4 DNA Ligase Buffer		*10x T4 DNA Ligase Buffer
Line 38:		Line 38:
	*Purified, linearized insert (likely in H2O or EB)		*Purified, linearized insert (likely in H2O or EB)

-	====Method====	+	===Method===
	#Mix reagents for a 10 μL reaction.		#Mix reagents for a 10 μL reaction.
	#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C		#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
Line 44:		Line 44:
	#Store at -20°C.		#Store at -20°C.

-	===Transformation===	+	==Transformation==
	Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].		Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].

-	====Reagents====	+	===Reagents===
	*[[Competent cells]]: XL2-Blue (Stratagene 200150).		*[[Competent cells]]: XL2-Blue (Stratagene 200150).
	**Learn how to make your own by [[Preparing_chemically_competent_cells]]		**Learn how to make your own by [[Preparing_chemically_competent_cells]]
Line 53:		Line 53:
	*ß-mercaptoethanol (ß-MAE)		*ß-mercaptoethanol (ß-MAE)

-	====Method====	+	===Method===

-	===Screening===	+	==Screening==
	Either a [[Restriction_digest\| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]]		Either a [[Restriction_digest\| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]]

-	==Critical steps==	+	=Critical steps=

-	==Troubleshooting==	+	=Troubleshooting=
-	==Notes==	+	=Notes=
-	==See Also==	+	=See Also=
	===Cloning Software===		===Cloning Software===
	{{#dpl:category=cloning\|category=software}}		{{#dpl:category=cloning\|category=software}}

Christopher C Vanlang: /* Procedure */

2012-01-13T00:58:42Z

Procedure

←Older revision		Revision as of 00:58, 13 January 2012
Line 25:		Line 25:
	===Enriching the insert===		===Enriching the insert===
	The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]		The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA\|purification methods]] and there exists several protocols for [[PCR]]
		+
		+	===Restriction Digest===
		+	Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].

	===Ligation===		===Ligation===
-	~~Just~~ see [[DNA ligation]]	+	The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].

	====Reagents====		====Reagents====

Christopher C Vanlang: /* Specific Protocols */

2011-12-30T03:52:32Z

Specific Protocols

←Older revision		Revision as of 03:52, 30 December 2011
Line 70:		Line 70:
	{{#dpl:category=cloning\|category=Protocol\|titlematch=%:%}}		{{#dpl:category=cloning\|category=Protocol\|titlematch=%:%}}

-	~~===Specific Protocols===~~	+
-	*[[Knight:TOPO_TA_cloning]]	+
-	*[[General_Cloning_Protocol]]	+
-	*[[Duffy:LIC_Cloning]]	+
-	*[[Gateway_cloning_system]]	+

	==Discussion==		==Discussion==

Christopher C Vanlang: /* Procedure */

2011-12-30T03:52:07Z

Procedure

Show changes

Christopher C Vanlang: /* Media and plates */

2011-12-30T03:15:06Z

Media and plates

←Older revision		Revision as of 03:15, 30 December 2011
Line 10:		Line 10:
	=== Media and plates ===		=== Media and plates ===
	1.2- Reagents		1.2- Reagents
-	*Tryptone: Fisher BP1421-500
-	*yeast extract: Fisher BP1422-500
-	*NaCl: Sigma S-3014
-	*MgCl2: ????
-	*glucose: ???
-	*KCl: ???
-	*Agar: DIFCO 214010
-	*DMSO: ????
-	*Ampicillin: Sigma A-9518
-	*Xgal (5-bromo--4-chloro-3-indolyl-ß-D-galactopyranoside): Sigma B4252-1G
-	*PGEM kit: Promega
	*[[LB]] (Luria-Bertami) agar plates		*[[LB]] (Luria-Bertami) agar plates
	*[[IPTG]] stock solution (100 nM)		*[[IPTG]] stock solution (100 nM)

Christopher C Vanlang: /* Media and plates */

2011-12-30T03:13:00Z

Media and plates

←Older revision		Revision as of 03:13, 30 December 2011
Line 21:		Line 21:
	*Xgal (5-bromo--4-chloro-3-indolyl-ß-D-galactopyranoside): Sigma B4252-1G		*Xgal (5-bromo--4-chloro-3-indolyl-ß-D-galactopyranoside): Sigma B4252-1G
	*PGEM kit: Promega		*PGEM kit: Promega
		+	*[[LB]] (Luria-Bertami) agar plates
		+	*[[IPTG]] stock solution (100 nM)
		+	*[[SOC]] medium
		+	*[[DYT]] medium

-	#Other consumables	+	*Other consumables
-	#*gloves	+	**gloves
-	#*sterile (???) plates	+	**sterile (???) plates
-	#*pH paper	+	**pH paper
-	#*pasteur pipettes	+	**pasteur pipettes
-	#*DW	+	**DW
-	~~#[[LB]] (Luria-Bertami) agar plates~~	+
-	~~#[[IPTG]] stock solution (100 nM)~~	+
-	~~#[[SOC]] medium~~	+
-	~~#[[DYT]] medium~~	+

	==Procedure==		==Procedure==