LAB 4:
Extraction of DNA from Bacteria
& Restriction Enzyme Digestion of DNA
EMB Plating Results
I. Objectives
The purpose of today’s lab is to learn the theory and practice of extracting DNA from bacterial
cells, to understand how restriction enzymes work, and to learn how DNA fragments can be
separated by size using gel electrophoresis. We also will examine the results of our EMB
plating to illustrate the use of an indicator media to identify a certain class of bacteria. By the
end of today’s lab, students should be able to:

Explain how DNA can be extracted from cells

Extract DNA from bacterial cells using a spin column procedure

Explain how restriction enzymes work and where they come from

Explain how agarose gel electrophoresis works

Predict how many fragments would be produced by restriction enzyme
digestion of genomes of various sizes

Set up a restriction enzyme digestion of DNA

Describe how EMB medium, and culture media in general, can be used to
identify bacteria
II. Safety considerations

WEAR GLOVES

Dispose of all chemical waste in the labeled containers

When handling plates with bacteria, leave the lids on and don’t touch colonies
III. Introduction
1. METHODS OF DNA EXTRACTION
Methods of DNA Extraction
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Several methods have been developed for extracting DNA from cells. Most of the methods
begin with lysing the cells and then separating out the DNA from other components within the
cell, such as proteins, carbohydrates, and lipids. Of particular importance is removing or
destroying the activity of nucleases (enzymes that breakdown DNA) that can degrade the DNA
post-lysis. The choice of method depends on several factors:
1. What the DNA will be used for downstream (PCR, transfection, sequencing)
2. Type of cells from which the DNA is being extracted (bacteria, plant tissue, blood,
hair)
3. The storage conditions of the DNA post-extraction (is a freezer available?)
4. Cost of the procedure
5. Toxicity of the procedure
Biologists are often interested in using the DNA as a starting template for the polymerase
chain reaction (PCR), which is one of the most widely used methods of analyzing DNA.
Fortunately, PCR can often be successfully performed on relatively “dirty” samples that contain
cellular debris in addition to DNA.
1. Chelex Extraction
Chelex® is an ion exchange resin that protects DNA from degradation by chelating the
polyvalent ions that are necessary cofactors for most nucleases. Its selectivity for divalent
cations (e.g. Mg2+) is 5,000-fold stronger than its affinity for monovalent cations (e.g. Na+).
Thus, it is extremely useful in the clean-up of heavy metal spills from waste water or industrial
accidents.
The Chelex extraction method is quite crude but is very quick and works well under many
circumstances. However, it can be unreliable, as its efficacy is affected by many variables,
including pH, ionic strength, and the strength of competing moieties. It is also not suitable for
extracting DNA from tissues like animal tissue, hair, and sperm, which contain resilient
proteins with disulfide linkages.
The method works best on cells that lyse easily, including soft tissue and bacteria. The resin is
stored as a 10% solution at 4
o
C and the extraction procedure begins with vortexing the cells
of interest with the resin. Typically, about 300 uL of 10% resin is used per reaction. A tube of
bacterial cells, a small piece of the tissue, or other source of DNA is added to the resin,
vortexed thoroughly, and then centrifuged (to ensure that the sample becomes embedded in
the resin). The tube is then incubated for about 20 minutes at 95
o
C, which causes the cells to
lyse and the proteins in the cells to denature. (Remember that most proteins are very heatsensitive and degrade readily at high temperatures.) After the incubation, the tubes are
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centrifuged and the supernatant (which contains a crude DNA fraction) is ready to use (see
the schematic below).
Add cells
Close lid,
vortex,
centrifuge
95 degrees C
20 minutes
Centrifuge
Chelex®
(10%)
Remove
Supernatant
into new tube
and retain
Chelex method for extracting DNA from cells or biological
swabs or stains.
2. Organic Extraction
Organic extraction is tedious, smelly, and toxic but produces high quality DNA and is very
reliable. It can be used on bacteria, blood, soft tissue, and even sperm, bone, or hair (as long
the cells and tissues are incubated with DTT and proteinase K beforehand to break down the
resilient proteins they contain).
Phenol/chloroform/isoamyl alcohol (PCA; 25:24:1) is added, in equal volume to the sample, to
solubilize the cell membranes and denature intracellular proteins. (Note: PCA can cause severe
chemical burns on contact with the skin.) The proteins and lipids are then separated from the
water-soluble DNA by centrifugation. The upper aqueous layer is retained and the lower
phenol layer is discarded.
Aqueous layer (clear)
Phenol layer (yellow)
Afterwards, the DNA is usually concentrated by ethanol precipitation. In a high-salt, 70%
ethanol solution, DNA precipitates, forming a stringy mass. It can then be removed from the
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surrounding solution by centrifugation. The supernatant is removed and the DNA is air-dried
and then re-hydrated with a small volume of water or Tris-EDTA buffer.
Some companies have modified the organic extraction method and replaced PCA with a salt
precipitation of the proteins. This procedure is less toxic and more convenient than the
traditional method and also works quite well.
3. Spin Column Extraction
Spin column extraction kits, such as Qiagen’s QIAamp® DNA Micro system, are becoming
increasingly popular because they yield high quality DNA without the messiness and toxicity of
organic extractions. Most methods lyse the cells with heat or lysis buffer (usually containing
SDS, a detergent). The column then takes advantage of the highly negatively charged nature
of DNA to separate the DNA from other cell components. The spin column membrane carries
positively charged silica groups that bind DNA very tightly in the presence of chaotropic salts,
which remove water from hydrated molecules in solution. Polysaccharides and proteins do not
adsorb to the membrane and are washed through. The DNA is then eluted from the
membrane under low (or no) salt conditions.
“Chaotropic” means chaos-forming. Chaotropic salts got their name from their ability to
disrupt the weak hydrogen bonds that normally hold water together and give it its loose,
pliable liquidity. In an aqueous environment, DNA is normally covered by a hydrate shell of
hydrogen-bonded water molecules, allowing it to
remain soluble. However, when chaotropic ions are
added, this hydrate shell is disrupted and the
immediate area around the DNA molecule becomes
hydrophobic. Under these conditions, the silica
membrane becomes the most suitable binding
partner for DNA, and it adheres to the DNA very
strongly.
QIAGEN QIAamp DNA Micro kits can be used to
extract DNA from all kinds of starting sources,
including blood, bacteria, tissue, eukaryotic ell
cultures, and swabs. One of the disadvantages
of the system is that chaotropic salts are
toxic and must be discarded as chemical
waste. However, the system has excellent yield
and the DNA can be used successfully for most
downstream applications, including PCR.
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QIAamp DNA procedure.
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2. RESTRICTION ENZYMES
Viruses called bacteriophages are major enemies of bacteria. These viruses infect bacteria by
injecting their own DNA into bacteria to force the bacteria to multiply the DNA. Bacteria have
responded by evolving a natural defense, called restriction enzymes (endonucleases), to cut
up and destroy the invading DNA. (Endonucleases attack DNA at internal sites in the molecule
rather than chewing it up from the ends the way exonucleases do.) A
restriction enzyme acts like molecular scissors, making cuts at specific
sequences of base pairs that it recognizes. They sit on DNA and slide along
the double helix until specific sequences of base pairs are recognized signaling the enzyme to stop sliding. The enzyme then cuts or chemically
separates the DNA molecule at that site, which is called a restriction site.
Bacteria prevent digestion of their own DNA by chemically modifying certain DNA bases within
the specific enzyme recognition sequence (usually by adding a methyl group to one of the
nitrogenous bases), which prevents the restriction enzymes from recognizing their sites. This
could be considered a very primitive immune system.
Restriction enzymes typically recognize palindromic sequences of base pairs - sequences that
have the same sequence when read 5' to 3' on each strand. An example of a 6 base-pair
palindromic sequence is given below.
5'-GAATTC-3'
3'-CTTAAG-5'
Some restriction enzymes may leave short unpaired nucleotide bases, called “sticky” ends, at
the DNA sites where they cut, whereas other restriction enzymes make a cut in the middle,
creating double stranded DNA fragments with “blunt” ends. Below is an example of a "sticky
cut." Note that when the DNA molecule is cut in this way, the ends of the resulting molecules
have unpaired nucleotides.
5'-G A A T T C-3'
3'-C T T A A G-5'
5'-G-3'
3'-C T T A A -5'
5'-A A T T C-3'
3'-G-5'
Example of a restriction enzyme cut resulting in "sticky," unpaired ends.
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In a blunt cut, there are no unpaired nucleotides because the enzyme cuts its restriction site
right in the middle.
5'-G A A T T C-3'
3'-C T T A A G-5'
5'-G A A -3'
3'-C T T -5'
5'- T T C-3'
3'- A A G-5'
Example of a restriction enzyme cut resulting in "blunt" ends.
If a specific restriction site occurs in more than one location on a DNA molecule, a restriction
enzyme will make a cut at each of those sites, resulting in multiple fragments. Therefore, if a
given linear piece of DNA is cut with a restriction enzyme whose specific recognition code is
found at two different locations on the DNA molecule, the result will be three fragments of
different lengths. If the given piece of DNA is circular and is cut with a restriction enzyme
whose specific recognition code is found at two different locations on the DNA molecule, the
result will be two fragments of different lengths. The length of each fragment will depend upon
the location of restriction sites on the DNA molecule.
One common restriction enzyme is Eco RI. This enzyme was isolated from E. coli (hence the
"Eco" in its name) and has the 6 base-pair recognition site 5'-GAATTC-3'. It cuts its sequence
as shown below.
Consider the two samples of DNA shown below.
Sample #1
5' - C A G T G A T C T C G A A T T C G C T A G T A A C G T T - 3'
3' - G T C A C T A G A G C T T A A G C G A T C A T T G C A A - 5'
Sample #2
5' - T C A T G A A T T C C T G G A A T T C G C A A A T G C A - 3'
3' - A G T A C T T A A G G A C C T T A A G C G T T T A C G T - 5'
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Question: If both samples are treated with the Eco R1 restriction enzyme, how many
fragments would be produced? Circle the recognition sites of the enzyme.
Like all enzymes, restriction enzymes function best under specific buffer and temperature
conditions. Eco RI functions best at 37
o
C (why?) and, when cutting DNA in vitro (outside of
the cell in a test tube), it prefers a reaction buffer that consists of various salts and other
reagents that hold the pH at 8.0.
3. GEL ELECTROPHORESIS
Gel electrophoresis is one of the most widely used techniques in molecular biology. It
enables researchers to separate out large biomolecules (DNA, RNA, proteins) according to
their size (number of nucleotides or number of amino acids). Electrophoresis refers to any
separation of molecules in an electrical field, and gel electrophoresis refers to the separation of
molecules in an electrical field in the presence of a supporting gel matrix. The matrix stabilizes
the system and also serves as a molecular sieve, allowing small molecules to pass through the
matrix more quickly than large ones.
In molecular biology, gels of agarose and polyacrylamide are usually used because the pore
size created by these matrices is in the right range for efficiently separating molecules the size
of nucleic acids and proteins. Agarose gels have larger pore sizes and therefore permit large
nucleic acids to enter the gel matrix and be separated. On the other hand, these gels have
fairly low resolution, and even very concentrated agarose gels (e.g. 2-3%) are limited to a
resolution of about 50 base-pairs. Polyacrylamide gels have pores that are too small to permit
large nucleic acids from entering the matrix. However, they are an excellent choice for
separating molecules of 600 nucleotides or less and their resolution is very high.
Polyacrylamide gels are used for DNA sequencing (which requires that DNA molecules differing
by a single base-pair be resolved) and are always used for the separation of proteins.
Three factors influence how rapidly a nucleic acid or protein will move through a given gel
matrix: (1) its mass (which is directly related to the number of nucleotides or amino acids in
the molecule, (2) its overall charge, and (3) its shape or conformation. The following rules
apply:
1.
All else being equal, less massive nucleic acids and proteins will move more quickly
through the gel than more massive ones.
2.
All else being equal, highly charged nucleic acids and proteins will move more quickly
through the gel than less highly charged ones.
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3.
All else being equal, nucleic acids and proteins with tighter conformations will move
more quickly through the gel than those with looser conformations.
Since there are three factors that influence migration rates, separating out molecules on the
basis of size alone can be problematical. In the case of double-stranded DNA, it’s relatively
easy because all DNA molecules are long, thin rods (i.e. have the same conformation) and
carry the same charge:mass ratio (one negative charge per nucleotide). Therefore, as long as
the gel is oriented such that the DNA molecules run through the gel from the negative to the
positive end, they will naturally separate out according to mass (length) alone.
Single-stranded DNA and RNA molecules tend to fold back upon themselves to form
complex secondary and tertiary structures (much like proteins). Thus, while all molecules have
the same charge:mass ratio, they can have quite different conformations. Therefore, such
molecules must be denatured as they are run by gel electrophoresis, ensuring that all the
molecules remain in a random coil conformation. This is usually achieved by the use of
formamide and/or urea.
The situation with proteins is even more complex because amino acids do not all carry the
same charge. Therefore, some proteins have an overall negative charge, some have an overall
positive charge, and some are neutrally charged. Proteins must therefore be treated with SDS
(a negatively charged detergent) prior to size separation analysis. SDS destroys the secondary
structure of the proteins and swamps them with negative charges. Thus, SDS forces all the
proteins into the same random coil conformation and charge:mass ratio.
SDS denatures proteins and swamps them with negative charges prior to gel electrophoresis.
http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html
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Agarose is a seaweed derivative and acts a lot like jello. If powdered agarose is mixed with
buffer and boiled, it enters solution. The gel is then cooled slightly and poured into a mold,
where it solidifies as it cools back to room temperature. Wells for loading the DNA samples are
created as the gel is poured by placing a well comb near one end. As the matrix cools, the well
comb will create holes in the gel, and very small volumes (usually less than 25 uL) of DNA
samples are placed in the wells. The gel is then submerged in a buffer, which carries the
electric charge and dissipates heat around the gel. When an electric charge is applied, the
molecules will begin to migrate, moving towards the positive electrode.
When the gel is removed from the electric field (the electrophoresis chamber), the molecules
will be present in a lane extending from the sample well, but they will not be visible. One way
to make nucleic acids visible is to treat the gel with an intercalating dye that fluoresces when
exposed to ultraviolet (uv) light. When a dye like ethidium bromide (EtBr) is used, the DNA
molecules of different sizes show up as orange fluorescent bands crossing the lanes. The size
of a piece of DNA of previously unknown size can be deduced by its position in the lane
compared to the position of standards of known size (a size ladder) that are also loaded on
the gel. The lower limit of detection on ethidium bromide stained agarose gels is about 10 ng
(double-stranded DNA).
Agarose gel electrophoresis of DNA. (1) Wells are created in the agarose slab by the gel comb as
the agarose cools and solidifies. (2-4) DNA samples are loaded into the wells sequentially using a
micropipettor. (5-6) When the power supply is turned on, the DNA samples move into the gel toward
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the anode
(+ end) and separate from one another according to size. During the actual gel run, the
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DNA fragments will not be visible, but they can be seen after ethidium bromide staining of the gel and
exposure of the gel to UV light. (From http://www.wikipedia.com/wiki/agarose+gel+electrophoresis)
When the DNA is loaded into the well on the gel, it is mixed with a loading dye. Two or three
negatively charged dyes are including in the loading dye, along with a heavy substance like
sucrose or glycerol. The dye serves two purposes. First, it moves through the gel in the same
direction as the DNA and is visible to the eye, so the progress of the electrophoresis can be
visualized and monitored despite the fact that the DNA in the gel cannot be seen until the gel
is stained and exposed to UV light. Second, it helps the DNA load into the well because the
loading dye is heavier than the gel buffer. Therefore, the DNA sample will "sink" into the well
of the gel and stay there until the current is applied.
4. RELATIVE SIZES OF GENOMES AND RESTRICTION ENZYME DIGESTIONS
Today, you will be extracting DNA from your bacteria cells and subjecting it to restriction
digestion with Eco RI. You will also be digesting the DNA from a bacteriophage called lambda.
(This DNA was purchased from a commercial source.) To predict the number of fragments you
would expect to generate when digesting these two genomes with Eco RI, you must know the
relative sizes of these two genomes as well as how often Eco RI's recognition site would be
found (on average) along a stretch of DNA. Let's do that now.
The DNA of lambda is approximately 48,000 bp. The E. coli genome is 100 times bigger at
approximately 4.6 x 106 bp. (The human genome is 1,000 times bigger still at approximately
3.3 x 109 bp.)
Since there are 4 bases in DNA and Eco RI's recognition site is 6 bases long, it would be
expected to cut (on average) about once every 46 bp = 4,096 bp.
Given this information, you can now calculate approximately how many fragments you would
generate by cutting each genome with a 6-base cutter by dividing the genome size by 4,096.
Question: How many fragments (on average) will you generate by digesting the lambda
genome with a restriction enzyme with a 6-bp recognition sequence? ___________
Question: How many fragments (on average) will you generate by digesting the E. coli
genome a restriction enzyme with a 6-bp recognition sequence? ___________
Question: What do you predict that you will see on a gel after running out these digested
products and staining with ethidium bromide?
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IV. Things to Do
PART A. EXTRACTION OF DNA FROM BACTERIA
1.
Put on a pair of gloves. If you have long hair, tie it back.
2.
Retrieve an overnight bacterial culture from the front bench. This is a culture that was
inoculated with cells from one of the colonies on your environmental plate.
3.
Using a micropipettor, transfer 1 mL of the culture to a 1.5-mL microcentrifuge tube.
Then return your overnight culture bottle to the front bench.
4.
Harvest the cells in the microcentrifuge tube by centrifuging the tube for 1 minute at
7,500 rpm in the microcentrifuge.
5.
Pull off the supernatant with a micropipettor (being careful not to disturb the pellet)
and discard the supernatant and tip in the Biohazard waste beaker at your bench.
6.
Resuspend the pellet in 180 uL of Buffer ATL with vigorous flicking or vortexing.
7.
Add 20 µL of proteinase K to the tube. Mix thoroughly by flicking and then incubate
at 56
o
C for 1 hour. Set your timer and vortex your tubes about once every 10
minutes during this incubation. (This will increase your DNA yield.)
While you are waiting for this incubation to go to completion, label your tubes
for Part B and then go on to Part C.
8.
Remove your tube from the 56
o
C water bath and vortex for 15 seconds. Then add
200 µL of Buffer AL to the sample and mix it well by vortexing.
9.
Add 200 µL of ethanol (96-100%) to the tube and mix it again thoroughly by
vortexing.
10.
Pipette the mixture from step 9 into a DNeasy Mini spin column. (Note that the column
is already contained within a 2-mL collection tube.) Then centrifuge the tube at 8,000
rpm for 1 minute. The original tube (now empty) should be discarded as
chemical waste.
11.
After centrifugation, discard the flow-through and collection tube as chemical
waste and save the column. (The DNA is now bound to the column.)
12.
Place the DNeasy Mini spin column in a new 2 mL collection tube and add 500 uL of
Buffer AW1. Centrifuge for 1 minute at 8,000 rpm and once again discard the
flow-through and collection tube as chemical waste.
13.
Place the column in a new 2-mL collection tube and add 500 uL of Buffer AW2.
Centrifuge for 3 minutes at 8,000 rpm to dry the DNeasy column membrane. This
time, you can safely discard the collection tube and flow-through in the trash.
14.
Place the DNeasy Mini spin column in a clean 1.5-mL centrifuge tube that has a lid.
Pipette 200 µL of Buffer AE directly onto the DNeasy membrane and incubate at room
temperature for 1 minute. During this incubation, the DNA will release from the
membrane and dissolve into the AE buffer.
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15.
Centrifuge the tube containing the column for 1 minute at 8,000 rpm. The DNA is
now in the flow-through, so do not discard it! Instead, discard the column in
as chemical waste and save the flow-through. Label the lid of the 1.5-mL tube
with your initials and section number. Your DNA is now ready for Part B.
NOTE: Due to the presence of the column in the tube, you will have to
centrifuge the tube with the lid open. If the lid pops off during the
centrifuging (which they sometimes do), simply transfer the flow-through to
a new 1.5-mL tube after the centrifuging is complete.
B. Set Up Restriction Digests
1.
Make sure you have the following items for setting up your restriction enzyme
digestions:

1 tube extracted DNA (from Part A)

1 tube lambda DNA

1 tube 10X Hind III buffer

1 tube sterile water

2 1.5-mL microcentrifuge tubes
The Hind III enzyme will be added to your tube by the instructor or TA when you are
ready. It must be kept on ice and is at the front bench. It is a 6-bp cutter with the
recognition site 5'-AAGCTT-3' and is made by the bacterium Haemophilus influenzae.
2.
Label your tubes "E" for E. coli and "L" for lambda. Also label your tubes with your
section number and initials so that you can identify them next time.
3.
4.
Set up your digestions as follows:
Tube
E
L
E. coli DNA
10 µL
--
Lambda DNA
--
10 µL
Hind III 10X buffer
2 µL
2 µL
Sterile water
7 uL
7 µL
Hind III enzyme
1 µL
1 µL
Gently mix the reaction by gently flicking your tube. Try not to introduce any bubbles
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as you do this. If you do introduce bubbles or splash the reaction up to the lid or into
droplets on the walls of the tube, a quick spin in the centrifuge will collect the sample
at the bottom of the tube.
5.
Place your tubes in the 37
o
C water bath for incubation. Your tubes will be incubated
for 2 hours to ensure that the digestion goes to completion. The instructor or TA will
remove your tubes for you and put them in the refrigerator until next time.
6.
When you are done, bring your tube of purified E. coli DNA to the front bench for
storage until next time. All other leftover reagents from the digestion can be put in the
trash.
C. EMB PLATING RESULTS
1. Retrieve the EMB plates that you streaked last time from the front bench. Record the
appearance of the colonies on the plate (morphology, color, etc.) and take a look at your
lab partner’s plates as well. What is the appearance of the colonies? Record your
observations in your lab notebook.
2. Examine the demo of E. coli and coliform bacteria grown on EMB. Do your colonies appear
to be coliform? E. coli? Record your conclusions.
V. LAB CLEAN-UP

Discard EMB plates in the large biohazard boxes.

Leave chemical waste beakers on the benches for removal and proper clean-up by the
lab technician.

All leftover tubes of buffer or other DNA extraction reagents should be discarded as
chemical waste.

All leftover tubes from the restriction enzyme digestion can be discarded in the trash,
as can your gloves.
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