Wheat Germ DNA Isolation
Darius Xavier Johnson
Purpose: The purpose of this experiment was to isolate the DNA of wheat germ cells and to analyze that DNA for absorption rates when denatured and annealed. Reagent Table:
Molecular Weight (g/mole)
Boiling Point (°C)
Skin and eye irritant
Skin and eye irritant
Introduction: Chromatin is a complex of DNA and proteins (histones) that make up the contents of the nucleus of a cell. It has 4 main functions: to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow for mitosis, to prevent DNA damage, and finally, to control gene expression and DNA replication. The structure of chromatin consists of a series of DNA wrapped around histones to form nucleosomes. But during mitosis the chromatin is compacted to form chromosomes. Each human has 23 pairs of chromosomes consisting of a sugar/phosphate backbone and nitrogenous base. There are 4 nitrogenous bases: Adenine, Guanine, Cytosine, and Thymine. Adenine and Thymine are always paired with each other while Guanine and Cytosine always paired. The other nucleic acid found in the nucleus is RNA. There are 4 major differences between DNA and RNA: DNA is double stranded while RNA is single stranded, RNA has Uracil instead of Thymine, DNA has a double helix structure, and RNA has a ribose for a sugar and DNA has a deoxyribose. In this lab we were focusing on DNA and its extraction from wheat germ. There are two to three basic steps in DNA extraction. The cell must be lysed to release the nucleus. The nucleus then must also be opened to release the DNA. At this point the DNA must be protected from enzymes that will degrade it. Once the DNA is released, it must then be precipitated in alcohol.
Procedure: This lab was spilt over 2 lab periods. During the first lab period the first step was to mix 50 ml of distilled water with a mixture containing dishwashing soap and salt in a beaker. The raw wheat germ was placed in a 50 ml conical tube. Enough soap and salt solution was added to the wheat germ to fill about 1/3 full. 0.1 ml of papain solution was then added to the wheat germ/soap/salt solution while stirring. The solution was stirred slowly for 5 minute and was allowed to settle for 2 minutes. A pipette was used to withdraw 1 dropper full of the clearer fluid. Fluid was added slowly to the 15 ml conical tube containing 10 ml of ice cold ethanol. The pointed end of the pipette was used to spool DNA. 1 ml of DNA was taken and placed in a 2 ml eppendorf tube. That concluded the first lab period. During the second lab period the tubes were spun in a microfuge and the liquid was drawn out and discarded. 2 ml of cold distilled water was added to the pellet and was spun again for a minute and decanted. Hot deionized water, about 2 ml worth, was added to the pellet and the DNA was vigorously resuspended using the vortexer. The pellet was allowed to cool to room temperature. The sample was then scanned using the spectrophotometer. The tube containing DNA was then heated in a boiling water bath for 5 minutes and the reading was taken again as quickly as possible.
Figure 1: This figure represents our DNA absorbance before heating at 260 and 280 nm
Figure 2: This figure represents our DNA absorbance after heating at 260 and 280 nm
Figure 3: This figure shows our DNA after it had been extracted from the wheat germ
Discussion: Hot water is used because the heat softens the phospholipids in the membranes that surround the cell and the nucleus. It also helps to denature the DNAase which would normally cut the DNA into small fragments that would not be visible. As the DNA denatures it begins to unravel and lose its shape thus rendering it inactive. But what exactly is wheat germ? It comes from...
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