DNA cloning protocols: Difference between revisions

From NESG Wiki
Jump to navigation Jump to search
mNo edit summary
No edit summary
Line 9: Line 9:
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”.&nbsp; <br>  
Multiple constructs (typically 2-6) are designed for each full length target and targeted domains within targets.&nbsp; Constructs are designed with <br>developing automated construct design software which employs homolog to the PDB, secondary structure predictions, and disorder predictions.&nbsp; <br>Precdicted singal peptides are removed from auto-generated constructs.&nbsp; 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.&nbsp; Predicted transmembrane helices are removed from auto-generated constructs.
 
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.  


<br>  
<br>  
Line 19: Line 17:
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>  
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>  


<br>
<br>  


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

Revision as of 15:33, 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 2-6) 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. Primers and vectors

Primers are designed using 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

  1. ↑ 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.
Retrieved from "https://nesgwiki.chem.buffalo.edu/index.php?title=DNA_cloning_protocols&oldid=2986"