Transformation of pGlO plasmid to an E.coli bacteria cell
We pipetted 250 ul of CaCl2 transformation solution to into two separate tubes. Next, we used a sterile loop to transfer two to four larger bacteria colonies from an E.coli plate into each of the two tubes. The bacteria was observed under UV light to make initial observations. We pipetted 10 ul of pGLO plasmid (0.08 ug/ul) into one of the tubes. Both tubes were incubated on ice for ten minutes. After ten minutes of incubation, the tubes were heat shocked at 42°C for exactly 50 seconds and then returned to ice. After two minutes on ice, we removed the tubes, pipetted 250 ul of LB broth to each tube, and then incubated them at room temperature for an additional 10 minutes.
We prepared four agar plates; two plates contained LB broth and ampicillin [LB/amp], another plate contained LB broth, ampicillin, and arabinose [LB/amp/ara], and the last plate contained only LB broth [LB]. We pipetted 100 ul of transformed bacterial suspension to the center of an LB/amp plate and then to the LB/amp/ara plate. Next, we pipetted 100 ul of untransformed bacterial suspension to the center of an LB/amp plate and the LB plate; these plates served as our control plates. We used a new sterile loop for each plate to spread the …show more content…
We began by examining the transformed bacteria on the LB/amp/ara plate and the LB/amp plate under UV light to find a single colony expressing the GFP gene. We obtained two culture tubes with 2 ml growth media and transferred the glowing colony from the LB/amp/ara plate into one tube and the non-glowing colony from the LB/amp plate into the other tube. Then, we shook both tubes vigorously by hand for thirty seconds, before placing them in a shaking incubator overnight at 32°C.
Bacterial Concentration and Lysis using
We pipetted 250 ul of CaCl2 transformation solution to into two separate tubes. Next, we used a sterile loop to transfer two to four larger bacteria colonies from an E.coli plate into each of the two tubes. The bacteria was observed under UV light to make initial observations. We pipetted 10 ul of pGLO plasmid (0.08 ug/ul) into one of the tubes. Both tubes were incubated on ice for ten minutes. After ten minutes of incubation, the tubes were heat shocked at 42°C for exactly 50 seconds and then returned to ice. After two minutes on ice, we removed the tubes, pipetted 250 ul of LB broth to each tube, and then incubated them at room temperature for an additional 10 minutes.
We prepared four agar plates; two plates contained LB broth and ampicillin [LB/amp], another plate contained LB broth, ampicillin, and arabinose [LB/amp/ara], and the last plate contained only LB broth [LB]. We pipetted 100 ul of transformed bacterial suspension to the center of an LB/amp plate and then to the LB/amp/ara plate. Next, we pipetted 100 ul of untransformed bacterial suspension to the center of an LB/amp plate and the LB plate; these plates served as our control plates. We used a new sterile loop for each plate to spread the …show more content…
We began by examining the transformed bacteria on the LB/amp/ara plate and the LB/amp plate under UV light to find a single colony expressing the GFP gene. We obtained two culture tubes with 2 ml growth media and transferred the glowing colony from the LB/amp/ara plate into one tube and the non-glowing colony from the LB/amp plate into the other tube. Then, we shook both tubes vigorously by hand for thirty seconds, before placing them in a shaking incubator overnight at 32°C.
Bacterial Concentration and Lysis using