Transcript
Justin Thompson
pGLO set-up Pre-lab
Describe the safety procedures in today’s lab. Make sure to include sterile techniques and handling of E. coli.
Your instructor will provide instructions, but these practices include, but are not limited to, the following:
• Decontaminating work surfaces once a day and after any spill of viable material with a 10% household bleach solution or ethanol.
• Decontaminating all contaminated liquid or solid wastes before disposal [This can be done in an autoclave (20 minutes at 121°C) or in a 10% bleach solution (soaked for 20 minutes).]
• Washing your hands after handling organisms containing recombinant DNA and before leaving the lab • Wearing protective eyewear and disposable gloves
• Not eating, drinking, applying cosmetics, or using personal electronic devices, such as laptops or cell phones, in the work area
What are plasmids?
a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes
What three genes are on the pGLO plasmid and what abilities do bacteria get from acquiring the pGLO plasmid?
a. Green Fluorescent Protein (GFP)- The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark.
b. beta-lactamase (bla)- which provides resistance to the antibiotic ampicillin, a member of the penicillin family.
araC- which allows for bacteria to utilize arabinose (sugar when glucose is not available in the environment.
What are the three main steps that we will do to introduce the pGLO plasmid into the E.coli cells?
Adding CaCl2 (Calcium Chloride - Transformation solution)
“Heat shocking” the cells
Incubating the cells in nutrient broth for a short time before plating them on agar
