- Describe the DNA within bacterial cells.
- Perform a DNA extraction and isolate a DNA molecule.
In this activity, you will extract a mass of DNA from bacterial cells visible to
the naked eye.
- The preparation of DNA from any cell type, bacterial or human, involves
the same general steps:
- Disrupting the cell (and nuclear membrane, if applicable),
- Removing proteins that entwine the DNA and other cell debris, and
- Doing a final purification.
- These steps can be accomplished in several different ways, but are
much simpler than expected. The method chosen generally depends
upon how pure the final DNA sample is and how accessible the
DNA is within the cell.
- Bacterial DNA is protected only by the cell wall and cell membrane;
there is no nuclear membrane as in eukaryotic cells. Therefore, the
membrane can be disrupted by using dishwashing detergent, which
dissolves the phospholipid membrane, just as detergent dissolves
fats from a frying pan. (The process of breaking open a cell is called
cell lysis.) As the cell membranes dissolve, the cell contents flow
out, forming a soup of nucleic acid, dissolved membranes, cell
proteins, and other cell contents, which is referred to as a cell
lysate. Additional treatment is required for cells with walls, such as
plant cells and bacterial cells that have thicker, more protective cell
walls (such as Gram-positive or acid-fast organisms). Additional
treatments may include enzymatic digestion of the cell wall or
physical disruption by means such as blending, sonication, or
- After cell lysis, the next step involves purifying the DNA by removing
proteins (histones) from the nucleic acid. Treatment with protein-digesting
enzymes (proteinases) and/or extractions with the organic solvent phenol
are 2 common methods of protein removal. Because proteins dissolve in
the solvent but DNA does not, and because the solvent and water do not
mix, the DNA can be physically separated from the solvent and proteins.
- In this activity, you will not attempt any DNA purification: your goal is
simply to see the DNA. You will lyse E. coli with detergent and layer a
small amount of alcohol on top of the cell lysate. Because DNA is insoluble
in alcohol, it will form a white, web-like mass (precipitate) where the
alcohol and water layers meet. Moving a glass rod up and down through
the layers allows you to collect the precipitated DNA. But this DNA is very
impure, mixed with cell debris and protein fibers.
- Before you begin the DNA isolation, make sure you know the procedure
to follow. Draw out a flow chart below including the amount of each
reagent and the time for that part of the procedure.
- Apply your PPE, including eye protection, for this lab. Locate the water
baths and the ice-cold ethanol. Determine a method for timing the various
- Label a 5-mL disposable tube and fill it with exactly 3 mL of distilled
water. Using a swab, inoculate E. coli from the stock culture and agitate
it in the 3 mL of distilled water.
- Add 3 mL of the detergent to the suspension of E.coli. Mix each tube by
- Place each tube into the water bath for 15 minutes.
Maintain the water bath temperature above 60°C but below 70°C. A temperature
higher than 60°C is needed to destroy the enzymes that degrade DNA.
- Cool the tube in an ice bath until it reaches room temperature.
- The next step involves precipitating the DNA by using solvent. Carefully
pipete 3 mL of ice-cold ethanol (it may be in the freezer) on top of the
detergent and E. coli suspension mixture. The alcohol should float on top
and not mix. (It will mix if you stir it or squirt it in too fast, so be careful.)
Water-soluble DNA is insoluble in alcohol and precipitates when it comes
in contact with it.
- By carefully placing a clean glass rod through the alcohol into the suspension,
a web-like mass will become evident; this mass is precipitated DNA.
The rod carries a little alcohol into the suspension, precipitating and
attaching to the DNA. Do not totally mix the 2 layers.