Dna Extraction Lab Report

The Amplification DNA Extraction from minced meat samples using the Polymerase Chain Reaction (PCR) and Gel Electrophoresis for Purification of the DNA.

Date: 14th/21st of October 2016
Partner(s):
Aisling Loughman.
Aim:
The aim of the experiment is learn the technique to extract DNA using minced meat samples (Pork, Beef and mixed), amplify the extracted DNA using the PCR Technique and further visualise the extracted DNA by Gel Electrophoresis under UV light.
Introduction:
“The method used for the amplification of DNA was developed back in 1985/1986 by a scientist named Kary Mullis, she was later awarded a Noble Prize in Chemistry for her work in 1993. The method she created has now become used widely in research, diagnostics
Vertical Electrophoresis is usually for the identification but Horizontal Electrophoresis is typically for DNA verification of DNA (this method will be demonstrated in the lab). The gel matrix are usually made up of one of two components. Polyacrylamide is used for proteins typically, it a made up of cross linked polymers of acrylamide and is quite toxic the other is Agarose which is a polysaccharide produced from red macro algae. The function of the gel is act like a molecular sieve slowing down the movement of the DNA in relation to their size and charge. The gel is a chemically inert substance which is not likely to interfere with the proteins or DNA as they move through the matrix which is essentially a web of pores. The agarose powder is to a conical flask with 1X TAE buffer and heated in the microwave until fully dissolved, it is important to ensure the powder is fully dissolved as spotting can occur in gel from lumps of undissolved powder. Once the gel is dissolved and cooled the gel is poured on to casting tray which has been prepared by double cello taping the sides of the cassette, once the gel is poured the combs are added to generate the wells. These wells are used to load the DNA samples when the gel had set. Multiple wells are manufactured in the gel so many samples can be loaded. The wells have other functions than just loading the samples, they mark the
The master mix we used already contained the loading dye and a constituent which increases the density. Once the gel is loaded the electric current is switched on so that the electrophoresis can be begin. The current runs from the negative electrode (cathode) to the positive electrode (anode) and because DNA and proteins are negatively charged they follow the same route this allows for samples to be pulled through the gel which separates. The rate at which the separation occurs depends on the size of the sample DNA. Shape also influences the rate of migration as globular molecules move faster than linear molecules. Small sample are dragged through the matrix quicker than larger fragments of DNA. SybrSafe is the stain used. It allows for the DNA fragments to be seen under U.V illumination. Ethidium Bromide is the main stain used as it intercalates with the DNA helix and like the SybrSafe it can be seen under U.V light. Ethidium Bromide is highly toxic and a known carcinogenic.” (Hearne,2016) (O’Meara, 2016)
Beef should be seen at 347 base pairs along the 100 base pairs ladder and pork should be seen at 267 base