DNA cloning protocols: Difference between revisions
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1. Target Selection. | 1. Target Selection. | ||
Protein targets are determined based on target selection criteria. [[Target selection]] page. | Protein targets are determined based on target selection criteria. See [[Target selection]] page. <br> | ||
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2. Construct design | 2. Construct design | ||
Revision as of 18:35, 2 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
Several constructs (typically 2-6) are designed for each target with the goal being to clone parts of the protein with a stable folded “core”.
The regions of the construct design is determined based on results from disorder and secondary structure predictions obtained from the DisMeta Server (www-nmr.cabm.rutgers.edu/bioinformatics/disorder)[1]. This server also predicts signal peptides, such as those from secreted proteins, and prediction of transmembrane regions, which will be excluded. In general, the N- and C-termini should not be located in the middle of a predicted helix of strand and disordered regions and the N- and C-termini are excluded.
3. Primers and vectors
Primers are designed using Primer Prim’er [2] 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
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