Biotechnology is the application of knowledge acquired from the study of living things. Scientists now understand what happens at each step of the complex biological and chemical process. These processes are carried out according to the instructions provided by each cell’s DNA. Genetic engineering is the process of isolating genes from one organism, building recombinant DNA molecule and inserting into another organism. Genetic engineering has been evident is agriculture, medicine, environment, and many other areas.
The following items will be required to complete this lab: a marker, two 2-mL sterile microfuge tubes, a microfuge tube rack, two sterile micropipets, 500 µl of 0.05 M CaCl2, one sterile inoculating loop, 10 µl of pUC18 solution, two Luria broth agar plates, two Luria broth agar with ampicillin plates, 500 µl of Luria broth, two spreading tools, clear tape, E. coli culture plate, ice bath, 42ºC water bath, and an incubator set at 37ºC.
1. Obtain two poured Luria broth agar plates. Label one of the plates “LB+,” and the other plate “LB-.”
2. Obtain two poured Luria broth agar with ampicillin plates. Label one of these plates “LB/Amp+,” and the other plate “LB/Amp-.”
3. Obtain two sterile 2-mL microfuge tubes. Label one of the tubes “+,” and the other tube “-.”
4. Using one sterile, plastic 1-mL micropipet, add 250 µl of ice cold 0.05 M CaCl2 to each of the sterile microfuge tubes.
5. Using a sterile, plastic inoculating loop, add 2-3 E. coli using colonies to each of the two 2-mL microfuge tubes. Try not to gouge the agar that the E. coli are growing on as no agar should be introduced to the tubes. Tap the inoculating loop firmly against the side of each tube to ensure that a colony is introduced to each tube. Discard the inoculating loop as directed by your teacher.
6. Use the micropipet from Step 4 to suspend the cells in the CaCl2 in the tubes. Do this by alternately drawing the solution from the tube into the micropipet and emptying it several times. Discard the micropipet as directed by your teacher.
7. Using a digital or fixed volume micropipet, introduce the antibiotic-resistance plasmid by adding 10 µl of pUC18 solution into the “+” tube, and mix the contents by tapping on the tube.
8. Place both microfuge tubes directly in the ice container for a total of 15 minutes.