Question

1. Why does the plasmid you are purifying have the beta-lactamase gene? 2. You are using a small column to bind DNA molecules during purification of the plasmid DNA. This column binds DNA via ionic interactions. What is the charge on the column which allows it to bind DNA? Answer positive" or "negative and explain your answer. 3. Where is the plasmid at the END of each of the following steps? Circle the letter of the correct answers for each step. Step #5 Breaking the cells (cell lysis) and precipitating the cell debris A. in the pellet B. in the supernatant Step #6 Binding the DNA to the column. A. Bound to the column B. In the solution that came out of the column into the microfuge tube. Step #7 Washing with Wash Solution A. Bound to the column B. In the solution which came out of the column into the microfuge tube Step #8 Washing again with Wash Solution A. Bound to the column B. In the solution which came out of the column into the microfuge tube. Step #9 Removing residual Wash Solution A. Bound to the column B. In the solution which came out of the column into the microfuge tube Step #10 Eluting the plasmid from the column A Bound to the column B. In the solution that came out of the column into the microfuge tube.

Answer 1

Ans-1 - The plasmid which is going to be purified has the ß. lactamase gene, because this go : because this gene acts as a marker gene. This gene provides antibiotic resistance to the host cell as it express the enzyme B. Lactamase which provide antibiotic resistance to the host cell by breaking the structure of antibiotics (only those antibiotics who have B. Lactam ring in their structure. Ans- 2- There is "positive" charge on the column which allows the DNA binding a via ionic interactions. Explanation DNA contains phosphoric acid in their structure and this each base pair of DNA possess - 2 unit of negative charge for the propes and strong ionic interaction, the column must contain positive charge. Ans-3- step-5- (6) In the supernatant. step-6- step-7- Bound to the column. ④ Bound to the column. Step 8- Bound to the column. Step-g - Removing residual Bound to the column. step-10 ® In the solution that came out of the column into the microfuge tube.