Beginning with the restriction digest of pARA and pKAN-R, briefly describe the steps required for constructing your recombinant plasmid from pARA and pKAN-R restriction fragments. Explain your answer including the manipulations of these two plasmids and their restriction fragments.
1. Combine 2.5x buffer with pARA & enzymes. Use pKAN-R, not pARA. When enzymes incubated, then will split to 2 parts each.
2. Heat tubes will denature enzymes with 5x buffer, ligase, & distilled water.
3. Control tubes come together & hydrogen bonds form at sticky ends of fragments. Ligase makes covalent bond stronger. The plasmid with rfp and ara genes will be used.