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Lab Exercise 8 - Extraction of Bacterial DNA
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Lab Exercise 8
Extraction of Bacterial DNA
Objectives:
1. Describe the DNA within bacterial cells.
2. Perform a DNA extraction isolating a DNA molecule.
Introduction
In this activity, you will extract a mass of DNA visible from
bacterial cells visible to the naked eye.
The preparation of DNA from any cell type, bacterial or human, involves the same
general steps:
(1) disrupting the cell (and nuclear membrane, if applicable),
(2) removing proteins that entwine the DNA and other cell debris, and
(3) doing a final purification.
These steps can be accomplished in several different ways, but are much
simpler than you probably thought. The method chosen generally depends upon how
pure the final DNA sample must be 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 that 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 grinding.
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 two 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.
Lab Exercise 8 - Extraction of Bacterial DNA
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.
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Materials:
disposable test tubes
deionized water
dishwashing detergent
(50% mixture)
glass rod
Stock Cultures:
E. coli
Water bath set at 60-70oC
ice bath
ice cold ethanol
Methods
1. 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 steps.
2. Label a 5ml disposable tube and fill it with exactly 3 mls of distilled water. Using a
swab inoculate E. coli from the stock culture and agitate it in the 3 mls of distilled
water.
3. Add 3 ml of the detergent to the suspension of E.coli. Mix each tube by gently
shaking.
4. Place each tube into the water bath for 15 min. Note: Maintain the water bath
temperature above 60oC but below 70oC. A temperature higher than 60oC is
needed to destroy the enzymes that degrade DNA.
5. Cool the tube in an ice bath until it reaches room temperature.
6. The next step involves precipitating the DNA by using solvent. Carefully pipetting
3ml 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.
7. 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 two layers.
Lab Exercise 8 - Extraction of Bacterial DNA
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Lab Exercise 8
Extraction of Bacterial DNA
Name___________
Lab _______
1. Why did the detergent affect the cell membrane allowing the
release of the DNA?
2. Consider the differences between your skin cells and bacterial cells. Also consider
the fact that you skin is an organ. Why did the detergent disrupt the bacterial cell
membrane but did not destroy our skin cells?
3. Describe the two locations of DNA in bacteria and explain the different functions of
each.
4. Complete the following table
Type of DNA
Length Purpose
Can the cell
Types of genes
survive without it? on this DNA.
Chromosomal DNA
Plasmid DNA
Date last updated 6/28/2017
©Janet Fulks