Experiment 2

Plasmid DNA Isolation, Restriction Digestion and Gel Electrophoresis

Plasmid DNA isolation introduction: diatomaceous earth. After RNaseA treatment,
The application of molecular biology techniques the DNA containing supernatant is bound to the
to the analysis of complex genomes depends on diatomaceous earth in a chaotropic buffer,
the ability to prepare pure plasmid DNA. Most often guanadine chloride or urea. The
plasmid DNA isolation techniques come in two chaotropic buffer will force the silica
flavors, simple - low quality DNA preparations (diatomaceous earth) to interact
and more complex, time-consuming high quality
DNA preparations. For many DNA manipulations
such as restriction enzyme analysis, subcloning
and agarose gel electrophoresis, the simple
methods are sufficient. The high quality
preparations are required for most DNA
sequencing, PCR manipulations, transformation hydrophobically with the DNA. Purification using
and other techniques. silica-technology is based on a simple bind-
wash-elute procedure. Nucleic acids are
The alkaline lysis preparation is the most adsorbed to the silica-gel membrane in the
commonly used method for isolating small presence of high concentrations of
amounts of plasmid DNA, often called minipreps. chaotropic salts, which remove water from
This method uses SDS as a weak detergent to hydrated molecules in solution. Polysaccharides
denature the cells in the presence of NaOH, and proteins do not adsorb and are removed.
which acts to hydrolyze the cell wall and other After a wash step, pure nucleic acids are eluted
cellular molecules. The high pH is neutralized by under low-salt conditions in small volumes, ready
the addition of potassium acetate. The for immediate use without further concentration.
potassium has an additional effect on the
sample. Potassium ions interact with the SDS The diatomaceous earth is centrifuged and
making the detergent insoluble. The SDS will contaminants are left behind in the supernatant.
easily precipitate and can be separated by DNA is eluted during incubation at 65oC and
centrifugation. In doing so the insoluble SDS traps then removed from the particles.
the larger genomic DNA and removes it from the
supernatant. This leaves the plasmid DNA and Restriction Digestion Analysis:
RNA in solution. The RNA is often removed by Restriction Digestion is the process of cutting
digestion with the addition of RNaseA. This DNA molecules into smaller pieces with special
leaves only proteins, carbohydrates and RNA enzymes called Restriction Endonucleases
nucleoside monomers in solution. A primary (sometimes just called Restriction Enzymes or
alcohol, such as ethanol or propanol is used to RE's). These special enzymes recognize specific
precipitate the DNA. This is accomplished by the sequences in the DNA molecule (for example
reordering of water, making the DNA aggregate GATATC) wherever that sequence occurs in the
and become insoluble. The result is a somewhat DNA.
pure pellet of DNA that can be resuspended in a
mildly buffered solution or water. Restriction Digests begin by mixing the DNA and
the RE, but it's unfortunately not quite as simple
Newer and more popular methods now exists as that. Restriction Enzymes are delicate and
that take advantage of the differences in need to be treated carefully. Because enzymes
physical properties of plasmid DNA and the are proteins and proteins denature as the
contaminants found in the alkaline lysis method. temperature is increased, RE's are always stored
Recently, a diatomaceous earth-based method in a freezer until they are used. In fact, all of the
was used to isolate the plasmid from cell lysate ingredients in a Restriction Digest are kept on ice
using the alkaline lysis method. This is also called until it's time for the reaction to begin. The actual
a siliga gel method. For this treatment the DNA reaction conditions vary from one enzyme to the
pellet is resuspended in RNaseA to remove the next, and include temperature, NaCl and/or
RNA by digestion. This is necessary because the MgCl2 concentration, pH, etc. All of these
RNA will compete with DNA for binding to the variables except temperature are optimized by

5
mixing the enzyme and DNA with a buffer gel electrophoresis. You should also be aware
specific for the enzyme of choice. Once all the that they do not necessarily migrate in the order
ingredients are mixed in the reaction tube, the presented in the figure above. Depending on
tube is incubated at the Restriction Enzyme's conditions the linear form may be faster than
optimal temperature for 1 hour or longer. Once supercoiled DNA or slower than nicked DNA.
the Restriction Digest is completed, agarose gel
electrophoresis is performed to separate the
digest fragments by size and visualize the
fragments and perhaps purify them for further
experiments.

Agarose Gel Electrophoresis

Linear
Mobility of a molecule under the influence of an
Supercoiled
electric field "is determined by its charge, its
formula weight, the pore size of the matrix
material and the strength of the electric field". Nicked
This holds true for the electrophoresis of DNA in
agarose gels. Because DNA and RNA have
constant anionic charge/mass ratios (one
phosphate for every nucleotide linkage) they
travel through the gel at a constant speed in
response to an electric current. The DNA
polymers of various sizes (sometimes called DNA Detection of Nucleic Acids in Gels:
fragments) are separated by the molecular The most popular method for the visualization of
sieving action of the pores in the agarose gel. double stranded DNA (or RNA) in agarose gels is
the fluorescent dye ethidium bromide (see
Agarose is used as the matrix in DNA below).
electrophoresis because it can be used to form NH2

much larger pores than polyacrylamide. The

lower the percentage of agarose or
polyacrylamide, the larger the pores.
Br-
N+
DNA migrates differently depending on whether H2N
it is circular or linear. Linear DNA migrates
CH2CH3

through the agarose pores in an end-on-end

fashion and snakes its way through the pores.
Circular supercoiled DNA is more compact and
migrates through the pores more easily. Linear
DNA almost always migrates more slowly than
circular supercoiled DNA. This dye is a planar molecule and intercalates
between the stacked base pairs of DNA. When
There are primarily two types of circular DNA, the dye becomes fixed in its position and enters
closed and nicked. Closed circular DNA has all the hydrophobic interior of the DNA, it becomes
of its nucleic acids linked with phosphodiester more fluorescent than free dye in solution. The
bonds and is supercoiled. Nicked circular DNA dye absorbs UV light at 300 to 360 nm and emits
has at least one broken phosphodiester linkage. light at 590 nm in the red-orange region of the
Nicked DNA is sometimes also called relaxed visible spectrum.
DNA because some of the tension present in
covalently closed and superhelically twisted Single-stranded DNA (or RNA) also will bind dye
DNA has been released. Closed circular DNA and can be detected by this method, but not
can be supercoiled. A single nick in one strand is nearly as well as double-stranded DNA. The
sufficient to release the tension caused by all affinity of the dye for single-stranded DNA is
degrees of supercoiling. relatively low and the fluorescent yield is poor.
Even though all of these DNA forms have the
same number of nucleotides and the same
formula weight, they migrate differently during
6
Electrophoresis standard curve:
The number of base pairs in a DNA (or RNA) 4) Add 200 µL of Cell Lysis Buffer and mix the
molecule is determined by comparison to the sample by inversion 10 times and incubate at
mobilities of standards. This method is only room temp for 5 min. DO NOT VORTEX! Vortexing
will result in shearing of the genomic DNA,
leaving free chromosomal fragments to
contaminate the plasmid DNA. This will result in a
co-purification of both DNA types and will ruin
any further experiments. The unlysed cells will be
very thick, almost like snot. The solution should
become slightly clearer if the cells have lysed.
Incubate for exactly 5 minutes, longer will lead
to inappropriate degradation of genomic DNA.
SDS solubilizes the phospholipid and protein
components. NaOH denatures the DNA as well
as proteins. Optimal lysis time allows maximal
accurate for linear DNA molecules because the
release of plasmid DNA from the cells without
molecular sieving properties of the gel are
release of genomic DNA. Long exposure to
determined by the end-to-end length of the
alkaline conditions may cause the plasmid DNA
DNA molecule and for the reasons discussed
to become irreversibly denatured. This DNA will
above. The distance migrated by several linear
run faster on a gel and is resistant to restriction
DNA molecules of a known number of base pairs
enzyme digestion.
is measured and a standard curve plotted as log
(base pairs) versus distance migrated. The
5) Add 200 µL of neutralization buffer and mix
number of base pairs in the unknown DNA
immediately but gently by inversion (5 – 6 times).
fragment is then determined by comparing the
After addition of this buffer the solution becomes
distance it migrated to the standard curve.
cloudy and very viscous. The lysate is neutralized
by the addition of acidic potassium acetate in
the neutralization buffer. The high salt (K+)
Experimental Procedures (DNA Isolation):
concentration causes the SDS to become
insoluble and precipitate. Plasmid DNA is too
1) Select a well-isolated colony for inoculation.
small to be trapped in the precipitate while
This will ensure maintenance of the plasmid and
genomic DNA and protein is. Any remaining SDS
that the plasmid is from a single cell. Inoculate
in solution will inhibit DNA binding to the column
into 5 mL of LB media containing 100 µg/mL
later so make certain that the solution is well
ampicillin and grow overnight at 37oC. This is
mixed.
best started in the morning the day before
starting the purification.
6) Mix the sample by inversion 5-6 more times
and centrifuge (max rpm in the microfuge) for 10
2) Transfer 1.5 mL of your cells to a 1.5 mL
min. at room temperature. Remove the
microfuge tubes. Harvest the cells by
supernatant immediately and place in a new
centrifugation (max rpm in the micro
sterile 1.5 ml tube. It is important that the
centrufuge) for 2 minutes at room temperature.
supernatant is clear and free of cellular debris.
Remove all traces of supernatant by inverting
The supernatant (cleared lysate) at this step
the centrifuge tube until all media has been
contains the plasmid DNA.
drained. The pellet can then be frozen at –20oC
for later use.
7) Prepare spin column. Attach a spin column to
a 10 mL sterile syringe barrel after removing the
3) Resuspend the bacterial pellet in 200 µL of
plunger. Mix the matrix by vortexing or repeated
buffer Cell Resuspension Buffer.
shaking and inversion of the bottle to insure that
Use a 200 μL pipettor and rinse the pellet in and
it is completely suspended. The silica has already
out of the pipet with the tip placed near or at
been equilibrated in binding buffer.
the bottom of the container. Go slowly; do not
aspirate the material into the pipet. Make sure
8) Add 1 mL of thoroughly suspended matrix to
that the bacterial pellet is completely
the cleared supernatant from the previous step
resuspended and no clumps remain.

7
and pipet up and down to mix. Pipet this mixture ¾ 10 mM EDTA 100 µg/ml
into your column/syringe from the previous step. ¾ DNase free RNase

9) Push the mixture through the mini column with Cell Lysis Buffer
the plunger. This will trap the plasmid DNA in the ¾ 200 mM NaOH
mini column. Remove the column, remove the ¾ 1% SDS store at room temp
plunger, and place the column back on the
syringe. Immediately wash the column with 2 mL Neutralization Solution
of wash solution by pushing it through the syringe ¾ 3.0 mM potassium acetate, pH 5.
barrel with the plunger.
Equilibration Buffer (Matrix storage buffer)
10) Complete the wash. Remove the spin ¾ 6 M guanidine hydrochloride.
column from the syringe barrel and place it in a
sterile 1.5 mL centrifuge tube. Place the Wash buffer
tube/column into the microfuge and spin for 2 ¾ 80% isopropanol diluted with water.
minutes to remove any residual traces of ¾ Ethanol can be used but is more expensive.
ethanol.
Experimental Procedures (Restriction Digests and
11) Elute the DNA from the matrix. Remove the Electrophoresis):
spin column and discard the microcentrifuge
tube. Place the column in a new 1.5 mL Setting up restriction digest.
microfuge tube. Add 50 µL of 65oC deionized You will need to get this part of the experiment
water. Promptly elute the DNA for 1 minute at going before you pour the gel as it takes
top speed. The decreased concentration of salts approximately 45 minutes of incubation time.
and increased temperature allow the DNA to be Setup the following in small eppendorf tubes:
solubilized and decreases the affinity to the
hydrophobic silica matrix. Save the eluted DNA. Tube DNA 10x EcoRI HindIII Water
Buffer
13) Determine the concentration of DNA in the L 5 μL -- -- -- 10 μL
sample. Blank the spec with 995 µL of water at E 5 μL 1.5 μL 1.5 μL -- 7 μL
260 nm and 280nm. Add 5 µL (exactly) of DNA H 5 μL 1.5 μL -- 1.5 μL 7 μL
to the 995 µL of water in the quartz cuvette. C 5 μL 1.5 μL 1.5 μL 1.5 μL 5.5 μL
Determine the absorbance at 260 and 280 nm. Plasmid 5 μL 1.5 μL 1.5 μL -- 7 μL
Given that double stranded DNA has an
absorbance at 260 nm of 1 for 50μg/mL, After you have set these tubes up, they will need
calculate the concentration of the DNA. The to incubate at 37oC for 45 minutes. λ /Hind
actual purity of the sample can be determined III/EcoRI DNA fragments will be used as the
by taking the ratio of absorbance at 260 to 280 standards for this experiment. Lambda DNA cut
nm. If the ratio is greater than 1.8 the absorption with HindIII/EcoRI consists of double stranded
is due to nucleic acids. A high quality prep DNA fragments with the following number of
should have a ratio of 2.0 to 1.8. If the ratio is basepairs: 21,226; 5148; 4973; 4268; 3530; 2027;
below 1.6 there may be proteins or other organic 1904; 1584; 1375; 947; 831; 564; and 125.
contaminates and the DNA can be extracted by
phenol/chloroform extraction for a final clean Gel Preparation
up. You will be provided with the following stock
solutions:
14) Analyze the sample by agarose minigel.
The class will run one minigel. Add 5 µL of DNA ¾ 10x TBE buffer/L: 108g Tris, 55g Boric acid,
Sample buffer to 10 µL of your pure DNA and 40 mL of 0.5M EDTA pH 8
load the whole sample. Include a DNA ladder by ¾ Agarose powder
adding 5 μL of sample buffer to 3 μL of DNA
marker. This apparatus was designed so that you could
use the buffer chamber as a gel-casting tray.
Buffers Place the gel tray in the chamber so that the
Cell Resuspension Buffer: black gaskets press against the walls of the unit.
¾ 50 mM Tris-Cl, pH 8.0 You will be using a 1% agarose solution in 1X TBE
8
buffer. Make sure the cap is loose on the Carefully slide the safety lid onto the unit. This will
agarose container and then heat the solution connect the power cords to the electrodes. Turn
until in the microwave until it starts to boil. on the power supply and run the gel at 115 volts
Remove the solution from the microwave and until tracking dye is almost to the bottom of the
swirl it to make sure the agarose has all dissolved. gel (~1hr).
Repeat the heating procedure until the solution
is completely clear. Let the solution cool to When the run is complete, turn off the power
about 50°C and then pour 40 mL of the warm supply, and slide off the safety lid. The casting
agarose into the tray. Insert the comb into its tray can be used as a convenient carrier for your
position at one end of the gel. Use the comb gel. Place the gel in a container and stain with a
with the fewest teeth. This particular apparatus is couple drops of 5 mg/mL ethidium bromide for
designed so that there is 1.0 mm agarose ~5 minutes. Place the gel and its carrier on the
between the bottom of the teeth and the base UV transilluminator and look at the gel through
of the gel. This insures that the sample wells are the safety cover. Be sure to wear goggles to
completely sealed when the gel has solidified. It protect your eyes from the UV light. Also, don't
will take about 15 min for the gel to solidify and expose your skin to the UV light too long or you'll
cool somewhat; it should appear uniformly get sunburn.
opalescent.
Results and discussion section of your lab report:
Plot the logarithm of the number of base pairs of
the standard (λ/Hind III/EcoRI DNA) on the y-axis
vs. distance migrated (x-axis). Do a linear
regression analysis and write the equation of the
line on the plot.

Include in report:

1. What was the size of your DNA fragments

(using your standard curve obtained from the
λ/Hind III/EcoRI DNA)? A nice table would be a
After the gel has solidified, lift the casting tray good idea here.
and place it in the buffer chamber. Pour in
enough buffer to completely cover the gel to a 2. What was the size of your plasmid DNA?
5 mm depth. It will take about 300 ml of 1x TBE
solution. Carefully remove the comb by lifting it 3. Be sure to attach the photograph of the gel
straight up. and indicate your partner’s name.

Preparing the samples:

You will make up your samples by adding 5 μL
tracking dye to each of your restriction digests.
The entire sample will be loaded onto the gel.
To fix a sample of your plasmid, take 5 μL of the
plasmid and add 5 μL of the tracking dye. Load
the entire sample.

Load onto the gel the following:

¾ Markers (λ DNA/HindIII/EcoRI)
¾ Uncut λ DNA
¾ EcoRI cut DNA
¾ Hind III cut λ DNA
¾ EcoRI/HindIII cut λ DNA
¾ Your uncut plasmid DNA

Using a micropipette, load all of each of your

samples into separate wells on the gel.