DNA cloning protocols: Difference between revisions
No edit summary |
mNo edit summary |
||
| Line 5: | Line 5: | ||
2. Construct design | 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”. <br> | 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”. <br> | ||
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)<ref>Paolo ref</ref>. 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. | The regions of the construct design is determined based on results from disorder and secondary structure predictions obtained from the [http://www-nmr.cabm.rutgers.edu/bioinformatics/disorder DisMeta] Server (www-nmr.cabm.rutgers.edu/bioinformatics/disorder)<ref>Paolo ref</ref>. 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 <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 derivatives (Clontech) 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 and to include the appropriate restriction enzyme cut site. The cut site will match with the corresponding multiple cloning sites in the vector. | 3. 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 derivatives (Clontech) 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 and to include the appropriate restriction enzyme cut site. The cut site will match with the corresponding multiple cloning sites in the vector. | ||
<br> | <br> | ||
== Typical University of Toronto Cloning Protocol == | == Typical University of Toronto Cloning Protocol == | ||
Revision as of 15:40, 20 November 2009
Typical Rutger's University Cloning Protocol for Target Proteins
1. Target Selection. Protein targets are determined based on target selection criteria. Link to 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 derivatives (Clontech) 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 and to include the appropriate restriction enzyme cut site. The cut site will match with the corresponding multiple cloning sites in the vector.
Typical University of Toronto Cloning Protocol
Coming soon...