Reporter or Chimeric Fusion Proteins - How to Make a Chimeric or Reporter Fusion Protein
Biochemistry, Molecular Biology, and Cell Biology Protocols >> Producing Reporter or Chimeric Fusion Proteins - How to Make a Chimeric or Reporter Fusion Protein
1. Make sure that your genes do not contain any of the restriction sites that you choose to cut out your gene with.
2. Perform PCR of the two genes that you wish to fuse together, removing the stop codon from the first gene. Remember to generate restriction sites on either site of each gene so that you can ligate it into a suitable vector.
3. Digest out the restriction sites from a vector, and purify out the vector to remove the DNA between the restriction sites. Also digest the introduced restriction sites from the 5' and 3' ends of the PCR products.
4. Perform a ligation with the genes produced by PCR and the target vector.
5. Transform the desired amount of ligated product into competent bacterial cells.
6. Select the desired colonies and grow up the bacteria in overnight cultures.
7. Purify the plasmid (eg can use commercial kits).
8. Store the purified plasmid at -80 degrees C or -20 degrees C in aliquots.
9. Express the subcloned fusion gene as a protein either in bacteria such as E. coli via induction, or in higher-level organisms such as mammals, where you can perform transfection.
10. If desired, purify the expressed fusion protein.