DNA cloning protocols: Difference between revisions
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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> | ||
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2. Construct design | 2. Construct design | ||
Multiple constructs (typically 2- | 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. | ||
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3. | 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> | |||
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Revision as of 15:42, 3 December 2009
Typical Rutger's University Cloning Protocol for Target Proteins
1. Target Selection.
Protein targets are determined based on target selection criteria. See Target selection page.
2. Construct design
Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets. Constructs are designed with
developing automated construct design software which employs homolog to the PDB, secondary structure predictions, and disorder predictions.
Precdicted singal peptides are removed from auto-generated constructs. If a target is predicted to be extra-cellular, only constructs with one
or no cystines are generated since extra-cellular proteins often require disulfide bonds for stability and propoer folding which are not readily
formed with the NESG's E. coli cytosolic expression systems. Predicted transmembrane helices are removed from auto-generated constructs.
3. PCR primers and vectors
PCR primers are designed using the NESG's PCR primer design software( Primer Prim’er [1] ) 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-FusionTM PCR Cloning System.
Typical University of Toronto Cloning Protocol
Coming soon...
References
- ↑ 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.