DNA cloning protocols

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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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