Lesson 3-3

DNA Isolation from Plant Tissue - Answer Key

After reading the procedure and performing the DNA extraction, answer the following questions.

1. What is the purpose of the following equipment or compounds:

Coffee grinder - breaks open the cell wall, plasma membrane and nuclear membrane to allow the DNA to come out into the solution.
Dry ice - freezes the cells and inactivates the enzymes that break down DNA, causes the tissue to shatter when ground with the coffee grinder.
CTAB buffer - a detergent, it breaks up and dissolves the lipid membranes and chemically alters the proteins and polysaccharides.
Heat bath - helps dissolve and break up lipids, helps denature proteins, inactivates enzymes.
Chloroform - a nonpolar solvent, the nonpolar (uncharged) molecules in the cell dissolve into it; helps denature proteins.
Centrifuge - separates the phases according to density.
Ice-cold isopropyl alcohol - a weakly polar solvent, DNA is not soluble in it; causes the DNA to precipitate out of solution when added to the aqueous supernatant.

2. Explain how the differences in polarity of the solvents (salt-water-CTAB solution, chloroform, isopropyl alcohol) allow this procedure to work.

The charged DNA molecules are initially dissolved in the polar salt-water CTAB solution. When this solution is mixed with chloroform, the nonpolar compounds in the mixture dissolved into the chloroform layer, away from the aqueous DNA layer. Last, when the weakly polar isopropyl alcohol is added to the aqueous solution, the large, negatively charged DNA molecules are no longer soluble in the solution, and they precipitate out as visible strands.

3. Given that animal cells don't have cell walls or as many polysaccharides in them, what might you do differently to isolate DNA from animal cells.

You wouldn't need to grind them in a grinder. A detergent is enough to break up the lipid plasma membrane.