DNA ligation & bacterial transformation 1. How DNA ligation works? Know the enzy
ID: 82929 • Letter: D
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DNA ligation & bacterial transformation 1. How DNA ligation works? Know the enzymatic reactions of T4 DNA ligase. 2. What type of reaction is catalyzed by DNA ligase? Why does it need ATP but not NAD^+? 3. Between what molecules/residues phosphodiester linkage is formed? Know the number & position of carbon atoms linked together with this bond. 4. Study the properties of T4 DNA ligase. 5. Why do we always keep enzymes and reagents on ice? 6. Know the importance of vector insert ratio in ligation reaction mixture. 7. Define Transformation. What is actually transferred during this process? 8. What is mean by 'competent' cells? 9. How competent cells are prepared? Why it is necessary to make competent cells for transformation? Understand the whole concept. 10. Why it is important to keep competent cells on ice until the heat-shock treatment? 11. What happens during heat-shock treatment? 12. Why the cells are incubated in SOC media for 45 min at 37 degree C after heat-Explanation / Answer
5) The enzymes and reagents are stored in the freezer to maintain their activity. Freezing temperature is the optimum temperature for these products for their proper functioning. Enzymes are stored in glycerol at -20 degree. So it will not get frozen completely and protects from denaturation.
7) Transformation is the process of genetic altration of bacaterial genome by introducing DNA from another cell or from a virus. Plasmids containing extrachromosomal DNA are used for the purpose of transformation.
8) Competent cells are E.coli cells which has been specially treated for the efficient transformation. They are more likely to incorporate foreign DNA if their cell walls are altered, there by DNA transfer can be carried out very easily.
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