The protocol we created was subject to change in order to produce optimal results.

1. Innoculate viable colony into a liquid culture
- Materials: LB media, dilution, micropipette and tips

2. Miniprep/Nanodrop
-Materials: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips

3. Digest
-Materials: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips

4. Gel
-Materials: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

5. Ligation

-Materials: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips

6. Transformation and Plate

-Materials: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips

7. Colony PCR (screening)

-Materials: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice

8. Gel

-Materials: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA