Producing A Flag-Tagged Protein

  1. Make sure that you’re interested in the gene sequence and gene sequences from Bendera tag does not contain a restriction site that you choose to ligate into the vector with.
  2. Perform PCR gene that you want to fuse with the Flag tag. If you attach a flag tag to the C-terminus of the protein, remember to remove the stop codon of the gene when designing your primary, and put a stop codon after the flag tag. If you attach a Flag tag to the N-terminus, it may be better to attach some codons downstream from the start codon rather than right after the start codon. primary ahead you normally would order of genes or DNA sequences, flag tag, and the reverse primer will usually be the order of Flag tag or your genes, respectively. Remember to generate a restriction site at one of the sites of the PCR products 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. After completing the PCR of Flag-tagged fused gene, also digested PCR products with restriction sites that have been designed at both ends of the gene.
  4. Perform ligation with PCR products containing the gene you are attached to the DNA sequence of the flag tag, 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. The flag-tagged gene express a protein both in bacteria such as E. coli via induction, or in higher organisms such as mammals, where you can perform transfection.
  10. If desired, the flag-tagged purifying the expressed proteins.

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