Genetic Engineering

PRACTICAL Plasmid Isolation Objectives 1. To extract and isolate a plasmid from a bacterial cell (E. coli). 2. To analyze the plasmids extracted by agarose gel electrophoresis. 3. To expose the student with the application of centrifuge and do an aseptic lab work. Methodology 1mL of culture was put in a 1.5mL microcentrifuge tube and was centrifuged at 10000rpm for 1 minute. ↓ Supernatant was discarded; pellet was resuspended with 150µL of Solution I, and then was vortex for 1 minute. ↓ 175µL of 0.4N natrium hydroxide and 175µL of 2% SDS was added to the suspension, then was inverted 5 times, followed by -20°C store for 5 minutes. ↓ 150µL of Solution II was added to the mixture then was inverted immediately; followed by another -20°C store for 5 minutes. ↓ Mixture was centrifuged at 10000rpm for 5 minutes, and then the clear supernatant was transferred to a new 1.5mL micro centrifuge tube. ↓ 600µL of cold isopropanol was added to the supernatant, then was vortex briefly, and was incubated in the room temperature for 2 minutes. ↓ The mixture was centrifuged at 12000rpm for 5 minutes to precipitate the DNA ↓ Supernatant was discarded; pellet was washed by adding 1mL of cold 70% ethanol then the tube was inverted several times. ↓ The mixture was centrifuged at 12000rpm for 1 minute and supernatant was discarded carefully ↓ The pellet was dry in the laminar air flow for 15 minutes then was resuspended with 70µL of Tris-EDTA (TE) buffer ↓ The plasmid extracted by agarose gel electrophoresis was analyzed (Gel electrophoresis was carried out on 0.8% agarose gel, at 250V, for 1.5 hours)

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Genetic Engineering

Discussion Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole. The plasmid was found in many different supercoiled forms in the bacteria. When plasmid was isolated from a bacterial culture, (in this case, E. coli) all the different supercoiled forms of the plasmid was isolated, and each will migrate differently on the gel, producing about three major bands and many minor bands. When this mixture of supercoiled plasmids is cut with a restriction enzyme, the different forms linearize and unwind. As a result they all become identical and run at the same rate, and usually only one band seen on the gel. Question 1. What is SDS? Explain its function in plasmid isolation. Sodium dodecyl sulphate (SDS) is a detergent; solubilised the phospholipids and protein component of the bacterial cell membrane, leading to lysis and release of cell content to obtain plasmid. 2. What are the functions of isopropanol and ethanol in plasmid isolation? Isopropanol is used to precipitate the DNA, but does not completely remove salts. Ethanol was added to ensure that. The ethanol-washed pellet contains the plasmid DNA, together with some RNA. 3. Plasmid is dissolved in TE buffer in the final step. Why? What is the function of Tris-EDTA (TE) buffer? TE buffer was added in the final step to remove residual salt, protein or RNA. Tris-EDTA (TE) buffer was used to keep DNA deprotonated (had one or more protons removed) and soluble in water. Conclusion Through this experiment, we become familiar with the aseptic technique to isolate the plasmid from bacterial cell; analyzing plasmid using agarose gel electrophoresis; and using the centrifuge. References http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html (140808)

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Genetic Engineering

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