- Plasmids can be available as expression plasmids or just for cloning
- A good plasmid length is between 4kb and 6kb
- The fluorescent protein can be inserted up or downstream of the structure (C or N terminus)
- The linker between both structures should be between 10 and 30 base pairs, but do not mess up the open reading frame of the construct (3 bases decode 1 amino acid -> linker should not shift the amino acid sequence of the second component)
- Good mutations for linkers are to alanine and glycine (nonpolar amino acids)
- MCS – multiple cloning site
- Choose restriction enzymes which require the same buffer then you can perform the digest at the same time.
Creating a plasmid with fluorescent protein attached to a structure of choice
- Find suitable plasmids: One plasmid with the structure (here: expression plasmid) and one plasmid with the fluorescent protein on Addgene OR order sequences via IDT or GeneArt
- Inspect both plasmids with SnapGene or VectorNTI for suitable restriction sites. Choose two unique restriction sites of the expression vector within the MCS.
- Cut out the fluorescent protein (FP) and add suitable restriction sites for the insertion of the FP into the MCS of the expression plasmid by PCR (design primers accordingly, i.e. with inserted restriction sites paired with the restriction sited at the MCS of the expression plasmid).
- Purify the PCR product.
- Digest the PCR product and the expression plasmid with the two chosen restriction enzymes.
- Ligate the open expression plasmid with the FP.
- Transform the final product into bacteria for plasmid replication. Pick a colony and do a MiniPrep.
- Test the plasmid by reverse digestion (control by running a gel of the end product).