Step 8. PCR of the Plasmid and Gel electrophoresis
After successful ligantion and transforation of pSB1C3 into our cells, we needed to check tosee if the plasmid contains our designed parts. For this we were required to design and use forward and reverse primers to run a PCR of the plasmid. This step id to ensure that the plasmid transformed into the cell are not empty plasmids but that they do indeed, contains our parts.
Using the primers we designed (see below), we ran a PCR of the plasmid to confirm the plasmid transformed into the cell are not empty plasmid but contains our gene.
After plasmid extraction from the bacteria (using the same protocl as step 2), a PCR was carried out followed by another gel electrophoresis run to confirm the presence of our plasmid.
The figure on the left shows the resulting gel electrophoresis run. The lanes that are empty are the empty plasmids while the lanes that have bands are the plasmids that have our gene successfully ligated onto them. The results of this test confirms our procedure was a success and the plasmid pSB1C3 contains our designed parts.
Step 9. Plasmid extraction, Drying and Submission to the iGEM Plate!.
Once we had determined the positive test for the presence of our designed parts within our sample, we could extract the DNA from the gel (using the same protocols outlined in step 5), we could then dry the plasmid and prepare for shipping using the iGEM shipping plate provided in the 2017 distribution kit. We are thrilled that in our first year studying synthetic biology we have successfully built a biobrick (see parts section).