During isolation of bacterial DNA, the mixture(cell debris, genetic material and protein in buffers) is transfered to a centrifuge tube and treated with an equal volume of chloroform.isoamyl alcohol(24:1 v/v) solution. This is then manually shaked for 15 minutes and centrifuge for 10 minutes at 3000rpm. Following centrifugation, 3 layers are observed in the tube; name the layers accordingly and state what is contained within them
The isolation of bacterial DNA involves a series of steps including breaking open the cells (lysis), removing proteins and other cellular components, and selectively precipitating DNA. One of the crucial steps in this process involves mixing the cellular material with a solution made of chloroform and isoamyl alcohol. By shaking this with the cell mixture and then centrifuging it, you divide the solution into different layers that are based on the density and solubility of the various components of the cell.
Following centrifugation, you would observe three phases (or layers). Name the layers accordingly and state what is contained within them:
1. Upper Layer (Aqueous Phase): This layer usually contains the water-soluble components, including the bacterial DNA. Because DNA is not soluble in the organic solvents used (chloroform and isoamyl alcohol), it remains in the water-based (aqueous) part of the solution, also known as the supernatant.
2. Middle Layer (Interphase): This is a thin layer that separates the aqueous and organic phases. The interphase contains proteins, lipids, and other cellular debris. After a long period of shaking, these cellular materials are separated from the DNA, because they are insoluble in water but soluble in the chloroform and isoamyl alcohol.
3. Lower Layer (Organic Phase): This contains chloroform and isoamyl alcohol along with proteins, lipids, and other cellular parts that are soluble in these organic solvents.
By layering out the cell mixture this way, scientists can selectively remove the bacterial DNA from the cell debris, lipids, and proteins. In the following steps of the DNA isolation protocol, the upper aqueous phase containing the DNA would be selectively removed and further refined to obtain pure DNA.
Remember, the principle behind this is based upon the fact that unlike other cellular components, DNA is not soluble in these organic solvents and remains in the aqueous phase. Also, DNA has a density that is less compared to the proteins and cellular debris, hence it floats on top. This combination of density and solubility characteristics allows for this crucial step in DNA isolation.
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