Week 9: Plating Kanamycin

 Introduction: 

    In lab this week, my partner Jessica Sabel and I worked on plating kanamycin with different concentrations onto TGY. Since our 96 wells plate results came back pretty decent, we decided to move onto plating the kanamycin. 

Fig. 1: Results

As shown here, the wells came out decent. Rows A-C (1-6) contains the kanamycin, TGY, and aquaticus. Row F contained the kanamycin and TGY (which is why there were exponential growth) and Row H only contained our blanks which were only TGY and our aquaticus. 

Fig. 2: Concentration Wells

Based on the results of the figure shown here, we came to the conclusion that the concentration of 1.5 ug/ml and 1.74 ug/ml are enough for aquaticus. 

Process: 

With that said, we laid our 10 plates of TGY which half will be the concentration of 1.5 ug/ml and the other half will be the concentration of 1.74 ug/ml. Before pouring the plates, we obtained two beakers of pure TGY and autoclaved each beakers for 30 minutes. After that, we calculating the amount of kanamycin and aquaticus going into each beaker. 1.5 ug/ml of 187.5 ul of kanamycin was added to Beaker 1 while 1.75 ul of 218.75 ul of kanamycin was added to Beaker 2. We mixed each of the beakers for a few minutes. We then started pouring each of the beakers to the plates (Again, Beaker 1 going to the first 5 plates and Beaker 2 going to the last 5 plates). We left the plates out to dry overnight until Jessica put the plates in the fridge the next morning using parafilms to compact them together. 

The next afternoon, Jessica, me, Kaylee, Jayce, and Christine all plated the aquaticus into the plates using a sterilized loop and a sterilized area. First, 100 ul of aquaticus was pipetted into the plate. We did this for all ten plates and then placed them into the incubator for 30 Degree Celsius. The plates will remain there for about 48 hours. 

Conclusion:

This process may or may not succeed or come out correct as we expected it to be. We may have to retry once more in order to get the correct and confident results in our plates. If the plates and procedure came out successful and correct, the MIC project will be finished and I will be moving ahead towards plasmid extractions. 

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