DNA cloning protocols - Revision history

Bs59 at 21:44, 10 December 2009

2009-12-10T21:44:09Z

← Older revision		Revision as of 21:44, 10 December 2009
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	== Typical Rutger's University Cloning Protocol for Target Proteins ==		== Typical Rutger's University Cloning Protocol for Target Proteins ==

	'''Target ~~Selection~~'''
			'''Target selection'''

	Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>		Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>

			<br>

	'''Construct design '''		'''Construct design '''

JohnEverett at 18:23, 3 December 2009

2009-12-03T18:23:37Z

← Older revision		Revision as of 18:23, 3 December 2009
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	'''Target Selection'''		'''Target Selection'''

	Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>		Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>



	'''Construct design '''		'''Construct design '''

JohnEverett at 18:23, 3 December 2009

2009-12-03T18:23:25Z

← Older revision		Revision as of 18:23, 3 December 2009
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	== Typical Rutger's University Cloning Protocol for Target Proteins ==		== Typical Rutger's University Cloning Protocol for Target Proteins ==

	'''Target Selection'''		'''Target Selection'''

	Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>		Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>

	'''Construct design '''		'''Construct design '''
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	'''PCR primers and vectors '''		'''PCR primers and vectors '''

	PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br> <br>
	<br>
	== References ==		== References ==

	<references />		<references />

JohnEverett at 18:23, 3 December 2009

2009-12-03T18:23:13Z

← Older revision		Revision as of 18:23, 3 December 2009
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	== Typical Rutger's University Cloning Protocol for Target Proteins ==		== Typical Rutger's University Cloning Protocol for Target Proteins ==

	1. Target Selection.		'''Target Selection'''

	Protein targets are ~~determined~~ based on target selection criteria. See [[Target selection]] page. <br>		Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>

	~~<br>~~		'''Construct design '''

	2. Construct design

	Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and proper folding that are not readily <br>formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.		Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and proper folding that are not readily <br>formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.
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	<br>		<br>

	3. PCR primers and vectors		'''PCR primers and vectors '''

	PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>

	<br>		<br>

	~~== Typical University of Toronto Cloning Protocol ==~~

	~~Coming soon...~~

	== References ==		== References ==

	<references />		<references />

TheresaRamelot at 18:11, 3 December 2009

2009-12-03T18:11:12Z

← Older revision		Revision as of 18:11, 3 December 2009
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	2. Construct design		2. Construct design

	Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and ~~propoer~~ folding ~~which~~ are not readily <br>formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.		Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and proper folding that are not readily <br>formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.

	<br>		<br>

TheresaRamelot at 18:10, 3 December 2009

2009-12-03T18:10:14Z

← Older revision		Revision as of 18:10, 3 December 2009
Line 9:		Line 9:
	2. Construct design		2. Construct design

	Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>~~Precdicted singal~~ peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no ~~cystines~~ are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.		Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.

	<br>		<br>
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	PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>

	<br>		<br>

	== Typical University of Toronto Cloning Protocol ==		== Typical University of Toronto Cloning Protocol ==

JohnEverett at 17:16, 3 December 2009

2009-12-03T17:16:15Z

← Older revision		Revision as of 17:16, 3 December 2009
Line 15:		Line 15:
	3. PCR primers and vectors		3. PCR primers and vectors

	PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and ~~carobxy~~ terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>

	<br>		<br>

JohnEverett at 15:54, 3 December 2009

2009-12-03T15:54:29Z

← Older revision		Revision as of 15:54, 3 December 2009
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	2. Construct design		2. Construct design

	Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs ~~homolog~~ to the PDB, secondary structure predictions, and disorder predictions.  <br>Precdicted singal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cystines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.		Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  <br>Precdicted singal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cystines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.

	<br>		<br>

JohnEverett at 15:52, 3 December 2009

2009-12-03T15:52:17Z

← Older revision		Revision as of 15:52, 3 December 2009
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	3. PCR primers and vectors		3. PCR primers and vectors

	PCR primers are designed using the NESG's PCR primer design software( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) ~~and cloned~~ into ~~either~~ pET15 or pET21 (Invitrogen) expression vector derivatives that have N- and C-~~terminal hexaHis~~ tags, respectively. ~~The~~ primers are designed to add 15 base pairs to the end of each PCR amplified fragment ~~that will match~~ with ~~the corresponding multiple~~ cloning ~~sites in the vector~~. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector <br>derivatives that have amino and carobxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment <br>desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>

	<br>		<br>

JohnEverett at 15:42, 3 December 2009

2009-12-03T15:42:45Z

← Older revision		Revision as of 15:42, 3 December 2009
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	Protein targets are determined based on target selection criteria. See [[Target selection]] page. <br>		Protein targets are determined based on target selection criteria. See [[Target selection]] page. <br>

			<br>

	2. Construct design		2. Construct design

	Multiple constructs (typically 2-6) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homolog to the PDB, secondary structure predictions, and disorder predictions.  <br>Precdicted singal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cystines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.		Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with <br>developing automated construct design software which employs homolog to the PDB, secondary structure predictions, and disorder predictions.  <br>Precdicted singal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one <br>or no cystines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily <br>formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.

	<br>		<br>

	3. ~~Primers~~ and vectors		3. PCR primers and vectors

	~~Primers~~ are designed using [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> and cloned into either pET15 or pET21 (Invitrogen) expression vector derivatives that have N- and C-terminal hexaHis tags, respectively. The primers are designed to add 15 base pairs to the end of each PCR amplified fragment that will match with the corresponding multiple cloning sites in the vector. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>		PCR primers are designed using the NESG's PCR primer design software( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) and cloned into either pET15 or pET21 (Invitrogen) expression vector derivatives that have N- and C-terminal hexaHis tags, respectively. The primers are designed to add 15 base pairs to the end of each PCR amplified fragment that will match with the corresponding multiple cloning sites in the vector. Fusion of the PCR product and the linearized vector is performed using the Clontech In-Fusion<sup>TM</sup> PCR Cloning System.<br>

	<br>		<br>