A scientist wants to produce a recombinant bacterial plasmid containing a gene for a fluorescent protein. The gene is initially obtained as mRNA from a donor organism. The plasmid vector is 4000 bp in length and contains a multiple cloning site with a single EcoRI site. The gene of interest is 720 bp long and is flanked by EcoRI sites. After the gene has been inserted into the plasmid, the recombinant plasmid is digested with EcoRI and the fragments are separated by gel electrophoresis. The results are shown in the table.
| Fragment length (bp) |
|---|
| 4000 |
| 720 |
Outline the steps involved in this process, from obtaining the gene to confirming its expression. Include the roles of reverse transcriptase, DNA polymerase (Taq), restriction endonucleases, DNA ligase, and a fluorescent marker.