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  • Cloning of MCP-tag Fusions in pMCP-tag(m) Vector (N9317)

    Protocol

    1. Cloning by PCR
      To subclone an appropriate cell surface signal peptide into pMCP-tag(m) Vector creating an N-Terminal fusion to the MCP-tag, use the available restriction sites SacI , SacII , NotI , EcoRV or HindIII which are located upstream of the MCP-tag. 

      To subclone the gene of interest into pMCP-tag(m) Vector creating a C-Terminal fusion to the MCP-tag, use the available restriction sites downstream of the MCP-tag: SbfI (PstI ), AscI, BamHI , XhoI , ApaI and KpnI

      Note: When making a C-Terminal fusion to the MCP-tag, note that there is a stop codon between the BamHI and XhoI sites, so SbfI (PstI), AscI, or BamHI must be used as the 5´ cloning site for your insert. 

      Primer Design and Cloning Hints:
      • Design your PCR primers to include a sufficient overlap with the sequence of the gene you want to amplify.
      • You may also want to include a stop codon at the C-Terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at that position.
      • For fusions upstream of the MCP-tag, ensure that a start codon is included. The addition of a Kozak sequence (e.g. GCCRCCATG, where the start codon is underlined) will increase the translation efficiency.
      • In general, any linker peptide between the proteins should be kept short to avoid degradation by proteases. If required, specific protease cleavage sites can be introduced into the linker peptide.
      • Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
      • Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.
      • After subcloning the gene of interest into pMCP-tag(m) Vector as a fusion with the MCP gene, the resulting plasmid can be used for transient expression of the MCP-tag fusion proteins in a suitable cell line.

    2. Direct Cloning
      Direct cloning can also be used to make fusions with the MCP-tag. This is only possible if the fusion partner has compatible sites adjacent to the gene of interest. 

      Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame. 

      Note: When making a C-Terminal fusion to the MCP-tag, note that there is a stop codon between the BamHI and XhoI sites, so SbfI (PstI), AscI, or BamHI must be used as the 5´ cloning site for your insert.

    3. Troubleshooting
      If subcloning of your gene of interest with the MCP-tag does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.). If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive control for the ligation reaction. 

      Alternatively try to subclone the MCP-tag gene into an expression vector already containing your gene of interest.