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  • Cloning of CLIP-tag Fusions in pCLIPf (N9215)

    Protocol

    1. Cloning by PCR
      To subclone the gene of interest into pCLIPf fused to the N-terminus of CLIPf, use the available restriction sites: NheI, EcoRV (blunt), AscI, SwaI (blunt), BsrGI, AgeI or EcoRI which are located upstream of the CLIP-tag.

      To subclone the gene of interest into pCLIPf fused to the C-terminus of CLIPf, use the available restriction sites downstream of the CLIP-tag: SbfI, BamHI, PmeI (blunt), XhoI, PacI or NotI.

      Note: When fusing the gene of interest to the C-terminus of CLIPf, note that there is a stop codon between the PacI and NotI sites, so SbfI, BamHI, PmeI, XhoI or PacI must be used as the 5´ cloning site for the insert.

      Note: PmeI and XhoI cannot be used together for cloning because they share a cytosine as part of their recognition sequences.

      Primer Design and Cloning Considerations:
      • Design the PCR primers to include a sufficient overlap (15–20 bp) with the sequence of the gene to be amplified.
      • For fusion to the C-terminus of the CLIP-tag, stop codon may be included at the C-terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at this position.
      • For fusions upstream of the CLIPf, ensure that a start codon is included. The addition of a Kozak sequence (e.g. GCCRCCATG, where the start codon is underlined) may 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 strategy 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 pCLIPf as a fusion with the CLIPf gene, the resulting plasmid can be used for stable or transient expression of the CLIP-tag fusion proteins in a suitable cell line.
      Direct Cloning
      Direct cloning can also be used to make fusions with the CLIP-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 fusing the gene of interest to the C-terminus of CLIPf, note that there is a stop codon between the PacI and NotI sites, so SbfI, BamHI, PmeI, XhoI or PacI must be used as the 5´ cloning site for the insert. 

      Note:
      PmeI and XhoI cannot be used together for cloning because they share a cytosine as part of their recognition sequences.

      Troubleshooting 

      Cloning of the Gene of Interest
      If subcloning of the gene of interest with the CLIP-tag does not work, reconfirm all the cloning steps (primer design, choice of restriction site, DNA isolation, ligation and transformation, etc.). If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive and negative control for the ligation reaction. 

      Alternatively, try to subclone the CLIPf gene into a mammalian expression vector already containing the gene of interest.