Question

BTH2732 Recombinant DNA Technology

Part A: Refer to Lecture 3 & Supplementary Video: https://www.youtube.com/watch?v SiwNtQYLKeU Virtual cloning of the Human p53 gene You are required to devise a set of practical methodologies in order to carry out a relatively simple molecular biology task. 1. You must identify the steps required 2. Formulate a set of practical protocols needed to carry out those steps . Consider all of the practicalities involved 4. Consider alternative procedures and how these would be carried out The desired outcomes: 1. Clone the human p53 gene sequence. 2. Identify the recombinant clone containing this sequence. 3. Isolate this clone Answer all the questions below 1. Describe p53 gene. What is the function of this gene in human? 2. What should be your starting material for this pro 3. How will you ensure that the bacteria retain the plasmid? 4. How do you isolate & purify the plasmid DNA from the bacterial host DNA? 5. How do you isolate & purify Genomic DNA to obtain the p53 gene from the Human ject? cells in culture? Explain the selection method to be used if the plasmid contains 2 antibiotic resistant genes 6.

For answer all questions below part, Ignore question 1. Just do question 2 and below

Answer 1

2. The starting material requires to be the gene to be cloned and the bacterial cell in which the gene has to be cloned. Also, isolation of the plasmid has to be done, into which the gene of interest is cloned.

3. In order to ensure that the gene has retained the plasmid, the gene of interest can be cloned close to the promoter of the plasmid, in the presence of nutrient elements such as lactose or glucose, to ensure that the gene is not on,y retained but also expressed.

4. Isolation of plasmid DNA can be done by different methods such as alkaline lysis method. In this method bacterial cells are grown followed by lysis of bacterial cells by Pelletier the cells in a micro centrifuge and the supernatant is removed. This is followed by phenol chloroform method, in which subsequent precipwtion of the plasmid occurs by the use of ethanol . When the bacterial solution is resuspended in the an isotonic solution . Ethanol addition will result in plasmid DNA precipitation and this is followed by centrifugation.

5. Isolation of p53 can be done by isolation of genomic DNA from the bacterial cell, followed by characterisation of the isolated plasmid, if it contains the genomic DNA.

6. If the plasmid contains two antibiotic resistance genes, then the bacterial colonies that have taken up the plasmid will grow on a plate containing the same antibiotics against which it is resistant. The colonies that do not grow, have not taken up the plasmid containing the gene for antibiotic resistance.