Introduction
The most essential method used in molecular biology is the extraction of protein, RNA, DNA, and biomolecules [Tan091]. There are a number of methods and technologies available for extracting DNA from cells. Generally, all these techniques involve four stages, namely disruption, lysis, removal of proteins and contaminants, and recovery of DNA. In some techniques, disruption and lysis are normally combined. The choice of a technique varies depending on the resources available, the specific downstream assay or the purity required for downstream applications, the number of cells or tissue available, and the number of samples in the study [San061].
The main purpose of this study was to extract DNA molecules from plant and animal cells using scientific procedures. DNA can be isolated from different types of cells. The initial step to do this usually involves breaking open the cell also referred to as lyse. Lyse is done by using a blender to grind a piece of tissue. The removal of proteins and other contaminants is done by adding a salt solution and a detergent solution containing sodiumdodecyl sulfate compound. The final step involves adding alcohol (ethanol or isopropyl) to the filtered lysate, which causes DNA to precipitate out of the solution as it is only soluble in water. The above-described process isolates the DNA from other cellular components, which was the main objective of this study.
Materials and Methods
Materials that were used to conduct the DNA extraction procedure included: a ziplock bag, measuring cylinder, DNA extraction buffer, chux cloth, funnel, conical flask, pipette, 15ml tube, ethanol, metal hook, Eppendorf tube, and ice. Materials that were used to conduct the Visualising DNA procedure included: a long wooden stick, microscope, nuclear (DNA) dye, and water. Materials that were used to conduct the controls procedure included: methylcellulose, a microscope, nuclear (DNA) dye, and water.
The method for extracting the DNA from strawberries was conducted as described below. First, the green sepals were removed from the strawberries and the sample was placed into a ziplock bag and sealed. The sample inside the bag was squished for 3 minutes using the fingers. Then, a measuring cylinder was used to measure 10 ml of DNA extraction buffer that was added to the sample. After adding the DNA extraction buffer, the mixture was further squished for 2 minutes and then filtered through a funnel and chux cloth into a conical flask. Using a transfer pipette, 3 ml of the sample was placed into a 15 ml tube and this tube was put on ice. The tube was labeled as strawberry and then 10 ml of cold ethanol was added to the tube and the tube was left on ice for about 10 minutes. Finally, a metal hook was used repeatedly to remove the isolated DNA from the tube and place it into a labeled Eppendorf tube.
The isolated DNA was visualized through a compound microscope using AGE. The procedure that was used was as follows: A sample of DNA was extracted from the tube using a long wooden stick and placed on a clean microscope slide. The DNA mass on the slide was leveled and stained with a common nuclear (DNA) dye. Little water was added and the coverslip mounted, and then observation of the DNA was made.
To determine whether the experimental results were legitimate, a controlled experiment was done through the following procedure. A small sample of methylcellulose (for positive control, Plasmid DNA was used) was added to a clean microscope slide. The mass on the slide was leveled and stained with a common nuclear (DNA) dye. Little water was added and the coverslip was mounted. Then, a small sample of the provided DNA control was added to another clean microscope. Its mass was leveled and stained with the dame common nuclear (DNA) dye. Water was added and the coverslip was mounted. Observations were made and recorded. The complete procedure described can be illustrated as shown in the diagram below:
Figure 1: DNA Extraction Process
Results:
Three procedures were conducted: one for the sample, one for the positive control, and the third one for the negative control. The results of the experiments were as shown in the following table:
Table 1: Slide Observations
- Positive Control
- Negative Control
- Sample Slide
- I could see the DNA clearly
- There was nothing on the slide
- I could see the DNA clearly
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Discussion:
According to the results, the experiment functioned as expected as the DNA was clearly visible in the Positive control experiment, not visible in the Negative control experiment, and clearly visible in the sample experiment. This also shows that all the reagents that were used worked properly. The detergent was used to disrupt the cell membranes in order to separate proteins and lipids by forming complexes with them. The DNA precipitated in the presence of alcohol because it is not soluble in alcohol rather it is soluble in water [Tan091]. Agarose gel electrophoresis separates DNA molecules according to charge, shape, and molecular size properties [Lee12].
The molecular weight marker is normally used when running a DNA gel to determine the size of the DNA fragments being tested [Sli13]. The DNA moved towards the anode in the electric field that is applied during the electrophoresis because its phosphate backbone is negatively charged. To visualize the DNA within the gel, ethidium bromide has to be added to the gel solution and the buffer [Adk96]. Ethidium bromide increases the fluorescence when it binds with the DNA and thus DNA bands can be viewed by exposing the gel to UV (ultraviolet) light [Adk96].
References list:
- Tan091: Tan & Yiap, 2009,
- San061: Santella, 2006,
- Lee12: , Lee, et al., 2012,
- Adk96: Adkins & Burmeister, 1996,
- Sli13: , Slish, 2013.
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