1. Plasmid extraction: ZYMO RESEARCH [1]

2. PCR: KOD/ Phusion Polymerase (EMD Chemicals Inc.,NJ) (Novagen Protocol)/ NEB Manufacturer’s kit

3. Restriction Digestion: New England Biolabs (Beverly, MA) Manufacturer’s kit

4. Ligation: Rapid DNA Ligase (Roche Applied Science, Indianapolis, IN). Manufacturer’s kit

5. Chemical competent cell and Transformation (Heat shock) (Protocol developed by hands on experience)

6. PCR product purification kit and gel extraction kit (ZYMO RESEARCH)