Week ten: The End!!

For my last week of the internship I tried my hand at cloning. I started by preforming a PCR on three samples, one from each bay. I did the samples in triplicates to make sure I had a full sampling of the bacterial community in them. After the PCR was preformed I pooled the samples and purified the PCR product before using a kit recombinant my amoA gene with a bacterial plasmid. I then used an electoporater to shock E. coli cells so that the plasmid could enter the cell and the E. coli could start producing the gene.  The plasmid also contained the lac Z operon as well as resistance to ampicillin.  I then made LB agar plates with ampicillin to prevent cells with the plasmid to grow and x-gal which will cause cells that have our gene in them to appear white and ones without the gene to look blue. I also learned in important lesson about making plates: if you do not add agar to the broth the plates will never harden.

After plating my cloned E. coli cells on the correct agar plates and letting them grow over night I picked colonies and re-streaked the on new plates so I could collet isolated colonies. Once the isolated colonies had grown I picked two from each plate and grew them in LB agar over night so the plasmid could be isolated. I also used the cells in a colony based PCR to test if the plasmid was successful integrated into the cell.

The next morning revealed minimal growth in the broth and when the cells were collected and the DNA was extracted concentration measured by the nanodrop was extremely low. The colony based PCR and an endonuclease reaction showed that none of the cells contained our designated plasmid. After examining the protocol we discover that the concentration of ampicillin that it told us to add to the plates were ten-fold less then the concentrated needed allowing contaminates to grow on the plates instead of our desired cloned cells.

Lydie will be making new plates and re-plating the cloned cells to rectify this mistake so we can correctly isolate our plasmids.

Overall it was a great summer and I learned more then I can express. I am excited to come back in September and start my masters program.