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 your genes.
  2. Perform PCR of two genes that you want to fuse together, remove the stop codon of the first gene. Remember to generate a restriction site at one of the sites of each gene so that you can ligate into a suitable vector.
  3. Digest exit restriction sites of the vector, and purify out the vector to remove the DNA between the restriction sites. Also digested restriction site was introduced on the 5 ‘and 3’ ends of the PCR product.
  4. Perform ligation with gene generated by PCR and target vectors.
  5. Transform the desired amount of product bound to the bacterial cells are competent.
  6. Select the desired colonies and grew the bacteria in a culture overnight.
  7. Purify plasmid (eg to use commercial kit).
  8. The purified plasmid Store at -80 ° C or -20 degrees C in aliquots.
  9. express the fusion gene subkloning as good protein in bacteria such as E. coli via induction, or in higher organisms such as mammals, where you can perform transfection.
  10. If desired, purifying the expressed fusion protein.