Introduction: The purpose of this experiment was to successfully transform pGLO plasmid into E.Coli cells. In the first segment of this laboratory exercise, one had to carry out a restriction digest. Restriction digestion is the process of cutting DNA molecules into smaller pieces with special enzymes such as, BamH1 and EcoR1. One had to determine which of the two plasmids, A or B, were pGLO or pWEB. A plasmid is a small circular DNA strand in the cytoplasm of a bacterium (Isite, 2013). In order to determine that, one had to use BamH1 and EcoR1 on two tubes each one with plasmid A and the other with plasmid B to observe the cuts made by each enzyme. The hypothesis for this segment of the laboratory exercise states that pGLO was plasmid B,
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Tube 1 and 2 contained buffer, water and plasmid A., however, in tube 1, one microliter of BamHI was added while, in tube 2, one microliter of EcoR1 was added to the solution. Tube 3 and 4 both contained buffer, water and plasmid B. In tube 3, one microliters of BamHI was added while one microliters of EcoRi was added to tube 4. All four tubes were placed in the centrifuge for approximately 30 seconds. The reactions were placed at 370 C for 30-60minutes.
Part2: Agarose Gel Electrophoresis
A solution with .75 grams of agarose was placed into a 75ml solution of 1X TAE in an Erlenmeyer flash and was swirled to mix. The solution was then heated in the microwave until the mixture started boiling and became transparent. Once the solution cooled to about 60oC ethidium bromide was added and swirled until completely dissolved. The mixture was later poured into the chamber. The gel was then left for approximately 10 to 15 minutes to solidify. Five microliters of loading dye was added to each of the PCR tubes. Once the gel was solidified, the PCR samples were added to the wells. The gel then runs at 100 volts for approximately 30 minutes. Once completed the gel was observed using a UV light box.
Part 3: Transformation
Two sterile microfuge tubes were obtained and were labeled +pGLO and –pGLO. Calcium Chloride was added to both microfuge tubes. A streak plate of E.coli was obtained, and 4-5