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Lab Report

DNA Plasmid Purification

Introduction

DNA Plasmid Purification is a technique which is used to separate plasmid DNA from bacteria E coli. The purpose of this purification is to make sure that the growth of the colonies on the vital centre point provides evidence that the cells are transformed. A plasmid exists in some bacteria as well as some organisms in a form of small circular non-chromosomal fragment of the DNA. Generally plasmid consists of one or more genes and these genes in the plasmid are responsible to display the important aspects as carried by the host bacteria. This helps the host cells in the survival in the toxic environment in the ampicillin resistant genes.

Majority of the plasmids contain at least one DNA sequence, and this DNA sequence act as the basis for the replication, therefore they have the ability to increase in number within the cell independently. The size of the plasmid always ranges from 1KB to less than 10Kb. The standard procedure to obtain purified plasmid DNA is by Electrophoresis by using agarose gel. The process is preferred because of its high speed. Therefore the purpose of this experiment is to demonstrate how DNA plasmid is purified from E coli cells.

Material and Method

0.8 agarose gel was obtained by reducing the concentration of 10x stock solution of TBE to 0.5x and was poured over 475ml of deionised water in 500ml measuring cylinder. Further addition of 25ml of the 10 x TBE buffer solution to make the solution up to 500ml in the cylinder. The parafilm is stretched over the top side so that the cylinder can be inverted without the leak of the liquid. Also taking into account that after putting one hand over the side it will be sticked to parafilm. The cylinder was then inverted from 2 to 3 times, 0.24 of the agarose gel was weighed into a conical flask of size 150-250ml and adding 30ml of 0.5x TBE buffer solution.

A cotton was than plugged on the top of the conical flask so that it can be heated in the microwave oven for 30 second under medium setting. After 30 seconds of heating the flask was swilled to decrease the overheating and the solution was checked for the presence of any solid particles. The mixture was allowed to cool at room temperature at 55°C so that the conical flask can be holded by hand. After the 30 µLof GelRed (a 1:1,000 dilution) was incorporated in the liquid the agarose and the flask were again swirled for thorough mixing.

The gel was then pored over the gel tray and the comb was inserted to make wells. It was then left for about half an hour for DNA plasmid purifying. The centrifugation of 2ml of bacterial culture was done under 8000 for 1 minute.

The supernatant medium was ignored and 3% Virkon solution was placed in the micro centrifuge tube that already contains the bacterial cell pellet placed in the tube rack. 250 µL of Buffer P1 was added and ...