DNA cloning protocols: Difference between revisions

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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.  Protein targets are determined based on target selection criteria.  Link to target selection page.


2. Construct design
'''Target selection'''
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)<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
Protein targets are selected based on target selection criteria. See [[Target selection]] page. <br>  
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.


<br>


== Typical University of Toronto Cloning Protocol  ==
'''Construct design '''


Coming soon...  
Multiple constructs (typically 2-3) 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 homology to the PDB, secondary structure predictions, and disorder predictions.&nbsp; <br>Predicted signal peptides are removed from auto-generated constructs.&nbsp; 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.&nbsp; Predicted transmembrane helices are removed from auto-generated constructs.  


== References ==
<br>
<references/>
 
'''PCR&nbsp;primers and vectors '''
 
PCR&nbsp;primers are designed using the NESG's PCR&nbsp;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>
 
== References ==
 
<references />

Latest revision as of 21:44, 10 December 2009

Typical Rutger's University Cloning Protocol for Target Proteins

Target selection

Protein targets are selected based on target selection criteria. See Target selection page.


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 homology to the PDB, secondary structure predictions, and disorder predictions. 
Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one
or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and proper folding that are not readily
formed with the NESG's E. coli cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.


PCR primers and vectors

PCR primers are designed using the NESG's PCR primer design software ( Primer Prim’er [1] ) for insertion into pET15 or pET21 (Invitrogen) expression vector
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
desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-FusionTM PCR Cloning System.

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.
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