To be able to make and agarose gel and perform gel electrophoresis in six different dyes. Also, to extract DNA from wheat germ.
Agarose gel is a substance that is used in science for gel electrophoresis and size exclusion chromatography. These processes use agarose gel to separate and analyze proteins and DNA. The medium is composed of a purified agarose powder that has been boiled in a buffer solution and then cooled into a gel. Agarose gel is most commonly associated with gel electrophoresis.
In this procedure, scientists use an electrical charge to move deoxyribonucleic acid, more commonly known as DNA, or ribonucleic acid (RNA) through a gel matrix toward a positive pole. Because the molecules have to move through small holes in the lattice bonds in the agarose gel, smaller molecules move much faster than larger molecules. Using ultraviolet imaging of the molecules’ movement and a formula that relates molecular weight to the speed of travel through the gel, scientists can determine the size of the molecules.
Electrophoresis is essentially a sieving process. The larger the fragment of DNA, the more easily will it become entangled in the matrix and, therefore, the more slowly will it migrate. Small fragments, therefore, run more quickly than large fragments at a rate proportional to their size. The relationship of size to migration rate is linear throughout most of the gel, except for the very largest fragments. Large fragments have a more difficult time penetrating the gel and their migration, therefore, does not have a linear relationship to size. The matrix can be adjusted, though, by increasing the concentration of agarose (tighter matrix) or by decreasing it (looser matrix). A standard 1% agarose gel can resolve DNA from 0.2 – 30 kb in length.
Agarose is extracted in the form of agar from several species of red marine algae, or seaweed, found in California and eastern Asia. Agar, a term derived from the Malay word agar-agar, meaning jelly, is typically derived from the Gelidium genera of seaweed. It is composed of both agarose and agaropectin molecules and provides support to the cell walls within the marine algae. Removed from the plant, the agarcan be used as a food thickener, much like gelatine, a laxative, or a medium for growing bacteria, fungus, or other microorganisms, when purified.
Part 2 – Principles and Practice of Agarose Gel Electrophoresis Question1: On what basis does agarose gel electrophoresis separate molecules? Answer 1: It separates molecules on the basis of charge, size, and shape.
Question 2: Explain migration according to charge.
Answer 2: When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.
Question 3: What conclusion can be drawn from the results of sample F? Answer 3: The conclusion that can be drawn is that the dye was moving the opposite direction of the flow because it is positively charged.
Question 4: Why is glycerol added to the sample solutions before they are loaded into the well? Answer 4: Glycerol is added so the sample you load is more viscous and won’t diffuse out of the well in the time it takes you to load the other samples before the current is turned on.
Question 5: What would happen if distilled water were substituted for buffer in either the chamber solution or the gel solution?
Answer 5: Water doesn’t conduct electricity well (i.e. the samples would not run properly), plus the buffer ensures that the solution remains at the proper pH so the DNA is evenly charged and remains annealed . So, if the current isn’t flowing well, the charge is not evenly distributed and the double strands are coming apart, you’d probably just end up with a smear of DNA on the gel and not the discrete bands you usually see.
Part 3 – DNA Extraction from Wheat Germ
Question1: What happens as the spooling rod is rotating in the aqueous- alcohol interface? Answer 1: The DNA starts slowly attaching itself to the spooling rod as it rotates in the test tube.
Question 2: Describe the appearance of the extracted DNA
Answer 2: The extracted DNA appeared like a thick web of strands of DNA on the spooling rod, the colour was rusty white; it looked like wet cotton.
Question3: List the four kinds of nucleotides of DNA and explain what differentiates them from one another and what composes a nucleotide. Answer 3: The four nucleotides in DNA are: adenine, thymine, guanine, and cytosine. Adenine pairs with thymine while guanine pairs with cytosine. Adenine and Guanine are purines, meaning that they are nitrogen bases composed of 2 rings. Hence, they are the larger bases. Cytosine and Thymine are pyrimidines, which are a group of nitrogen bases with only one ring. Therefore, they are smaller than purines. 5-carbon sugar, a phosphate group, and a nitrogenous base composes a nucleotide.
Question 4: What is the function of DNA in the cells?
Answer 4: DNA does not just “help cells reproduce” it is the genetic blueprint for every living being. DNA basically stores info and tells the cell what to do. All of your genes are in your DNA.
The DNA denaturation which causes smear occurs because, whenever a whole plant genomic is cleaved with an enzyme, usually a smear of DNA fragments can be seen on agarose gel after electrophoresis. And normally, clear, discrete bands appears on the background of smear. In this lab dyes were used and the mixed dye was the only one that separated into three different colours. Therefore, it is noticeable that when electrophoresis are being conducted, smaller DNA fragments travels at a faster rate and larger DNA fragments travel at lower rate.
We learned a great deal from the completion of the extractions. Besides the thrill of observing DNA, the genetic messenger of life, in its visible form, we also gained an excellent knowledge of the basic procedures and techniques for extraction of DNA from cells. While we were not able to observe the results of the addition of some substances (for instance, the detergent that was intended to solubilise the cellular and nuclear membranes), we did observe the success of later steps that would most likely indicate our hypotheses had been correct (for instance, when the ethanol precipitated the DNA out of the wheat germ solution, it was an indication that we must have been correct in our detergent hypothesis plasma. Above all, we gained invaluable experience with the essential heart of DNA technology: the extraction of the nucleic acids from cells.