Cloning of CLIP-tag Fusions in pCLIP-tag(m) (N9211)
Protocol
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Cloning by PCR
To subclone the gene of interest into pCLIP-tag(m) fused to the N-Terminus of the CLIP-tag use the available restriction sites ClaI, EcoRV (blunt) and EcoRI which are located upstream of the CLIP-tag.
To subclone the gene of interest into pCLIP-tag(m) fused to the C-Terminus of the CLIP-tag use the available restriction sites downstream of the CLIP-tag: SbfI, AscI, BamHI, XhoI and NotI.
Note: When fusing the gene of interest to the C-Terminus of the CLIP-tag, note that there is a stop codon between the XhoI and NotI sites, so SbfI, AscI, BamHI or XhoI must be used as the 5´ cloning site for your insert.
Primer Design and Cloning Considerations:- 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 CLIP-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 pCLIP-tag(m) as a fusion with the clip10m 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 the CLIP-tag, note that there is a stop codon between the XhoI and NotI sites, so SbfI, AscI, BamHI or XhoI must be used as the 5´ cloning site for your insert. - Troubleshooting
If subcloning of your gene of interest with the CLIP-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 re-try the cloning using different restriction sites. Be sure to include a positive control for the ligation reaction.
Alternatively try to subclone the CLIP-tag gene into an expression vector already containing your gene of interest.
