Construction of I1 to I3

5 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.

I1 I2

I2 I3

Selected colonies were inoculated overnight for getting enough material for doing plasmid extraction.Glycerol stocks were made before performing plasmid extraction. After extracting the plasmids, the concentration was measured. The plasmids were digested with the appropriate enzymes as per the flowchart and purified to remove the enzymes. The correct parts were ligated using a Tetracycline backbone.

Unfortunately the next day we did not get any colonies for all the 3 plates. It seems that the Tetracycline plates were not effective. As we were not sure about the effectiveness of the Tetracycline plates, we did the ligation and transformation again using Tetracycline backbone and also another replicate using Chloramphenicol backbone.

Colonies were obtained only for the Chloramphenicol backbone transformations. Hence, we decided to go ahead with using these colonies itself. However, our initial plan was upset and we would not end up with C3 backbone for the final constructs. At this time we felt the need to have more antibiotic resistance backbones to have more flexibility in our plan and overcome such problems. We were not able to obtain any colonies with Tetracycline plates until the end of the project.