DNA Digestion and Ligation

1 Mar 2022

Losel Baro, Nikita Damyan, and Zhelah Guzeh

Introduction:

We were selected for the Bacteria and Disease research stream at Minnesota State University Mankato, led by Dr. Yongtao Zhu. The purpose of our stream is to delete the sprB motility gene in Flavobacterium psychrophilum using the pYT313 plasmid. F. psychrophilum causes bacterial cold-water disease in salmonoid fish. We want to know if the motility of this bacterium is related to its ability to cause disease. In order to achieve this goal, we will insert the upstream and downstream regions of sprB to the plasmid pYT313 first.

Specific Content:

Over the past couple of weeks, we have worked on pipetting, serial dilution, plasmid preparation, DNA electrophoresis, media preparation, and transformation.

Figure 1. Zhelah is preparing the centrifugation for DNA purification.

We performed digestion on our purified plasmid and upstream region of sprB obtained by PCR. Digestion was done with specific DNA restriction enzymes which were BamH1 and Sal1 to generate sticky ends on the DNA, required for the next step – ligation.

Prior to our ligation process, we tested our products using a nanodrop machine to look at the concentration and to see how pure they were. Our target was 200 nanograms/microliters, but there was contamination in the plasmid due to an unknown problem since we did not reach our targeted number. The purpose of the ligation process was to insert our upstream region into our digested plasmid. We did not experience any problems during our ligation.


Figure 2. Nikita is measuring the concentration of the pYT313 plasmid.

Conclusion:

We have had very minor problems, but we were able to obtain the results that we needed to move forward. We performed the transformation using our ligation product and Escherichia coli cells. We created 11 LB Agar plates laced with ampicillin then, we transferred our transformation products onto two of the plates to begin the colonies’ growth. Both of our plates did not show significant growth so, our plan is to replate our transformation products to grow more colonies as well as perform a PCR using the colonies.

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