ChE 170L Laboratory
Exercise 3. Isolation of Plasmid DNA
Objectives
From this exercise, the structural differences between plasmid DNA and bacterial
chromosomal DNA should be understood. These differences are used to separate plasmids from
chromosomal DNA and other debris in a cell culture of E. coli. Various methods, both physical
and chemical, are utilized throughout the plasmid isolation process. Students should realize the
importance of each step as it pertains to the overall isolation scheme. The method of gel
electrophoresis will also be introduced as a technique for separating DNA for visualization by
staining with ethidium bromide.
PreLab Questions 2
Please limit your answers to a few sentences IN YOUR LAB NOTEBOOK
1. Under the conditions of this experiment, what happens chemically to cause the
denaturation of chromosomal DNA? What phenomenon helps to prevent the
denaturation of plasmid DNA?
2. What chemical detergent is used to precipitate out chromosomal DNA and high MW
RNA? What effect(s) does this solution have in this experiment?
3. In this plasmid preparation procedure high and low MW RNA are precipitated out and
discarded. Why then, do we still add RNase in the final suspension?
4. In what direction does the DNA run along the electrophoresis gel? Why?
5. Ethidium Bromide is a powerful mutagen (extremely carcinogenic) that may be fatal if
inhaled and harmful if swallowed or absorbed through skin. It causes irritation to skin,
eyes, and respiratory tract, and may cause heritable genetic damage. What precautions
should you take while working with EtBr in this lab?
Background
Plasmids exist in bacterial cells as circular DNA molecules and can range in length from
1000 to 200,000 base pairs. In nature, plasmids evolved as harbingers of recombinant DNA.
Some plasmids naturally carry genes that afford resistance to antibiotics and can provide a
selective advantage to host cells. In a process called conjugation, plasmids can be physically
passed from one cell to another, thus conferring the drug resistance to future generations.
Several plasmids, such as pBR322 (an ancestor to pUC19), have been extensively characterized
by molecular biologists. Plasmid pBR322 contains a gene for tetracycline resistance and a gene
for ampicillin resistance. This information can be used very simply to separate cells that contain
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the plasmid from those that do not. Only cells containing the pBR322 plasmid will grow in a
medium containing either of these antibiotics.
Plasmids are invaluable tools in genetic engineering. Well-characterized plasmids are
applied as cloning vectors, or vehicles for the insertion of foreign DNA into a bacterial host.
Entire genes or sets of genes can be spliced onto a plasmid, thus converting host cells into
protein factories when the DNA blueprint is translated via cellular machinery. They are
autonomous from the cell’s chromosomal DNA in that they can be replicated independently of
the replication of genomic DNA. This replication can be controlled such that the copy number
can be increased to over 1000 plasmids per cell. At this point the cell expends substantial energy
into expressing the gene(s) of interest and producing the protein(s) desired by the genetic
engineer.
Central to the task of plasmid manipulation is isolation from its bacterial source.
Fortunately, plasmid DNA and cellular chromosomal DNA are physicochemically distinct
enough to yield relatively simple procedures for plasmid separation. Conditions can be imposed
which cause chromosomal DNA to be irreversibly denatured due to disruption of the hydrogen
bonds involved in DNA base-pairing. Under these same conditions, the strands of circular
plasmid DNA are tightly intertwined and are unable to separate from each other. This
phenomenon, called supercoiling, can be illustrated with a simple demonstration. Suspend a
shoe by its laces, then spin the shoe until it stops. Double helix “DNA” results. Now support the
shoe, still holding the laces, and continue to twist the shoe. Tension in the laces causes the
double helix to wind around itself, yielding “supercoiled DNA.” Supercoiling renders plasmid
DNA far less susceptible to denaturation than the chromosomal type (Cantor and Schimmel).
Plasmids will be isolated from the host cells using a commercially available isolation kit
(QIAGEN) that relies on the principles of the modified alkaline lysis method of Birnboim and
Doly, and on the binding nature of DNA and a silica-gel membrane. Whole cells, derived from a
single transformed colony, are pelleted in a centrifuge and re-suspended in a small amount of
buffer. This buffer also contains ribonuclease A (RNase A), an enzyme that breaks down and
separates contaminant RNA from the plasmid DNA. Then at high pH, and with detergent
present, cell lysis is induced. A third, high salt solution is added to precipitate out the denatured
chromosomal DNA and the insoluble salt of the detergent used, as well as remaining cell debris.
This precipitate is pelleted out by centrifugation and discarded, while the supernatant is added to
a column which contains a silica-gel membrane. The high salt condition induces the binding of
DNA to the membrane while other materials present in the supernatant are washed through with
centrifugation. The column is washed with a solution that contains a large amount of ethanol, to
further remove unwanted materials. Finally, a low salt buffer is added to the column that induces
the dissociation of the DNA and the silica-gel. One last centrifugation produces a solution of
intact plasmid DNA in buffer.
DNA can be viewed and DNA fragment sizes can be deduced from gel electrophoresis.
This invaluable technique separates fragments based on molecular weight. The DNA is loaded
into wells at one end of the gel, and a charge gradient is imposed across the gel matrix. The
DNA backbone consists of negatively charged phosphate groups, so all fragments migrate in
response to the applied voltage. Mobility through the gel matrix is limited by the diffusivity of
the DNA in the gel pores. Larger molecules have a harder time getting through the pores than do
smaller ones, so small DNA fragments will move quickly through the gel while large ones stay
behind. The molecular weight of each unknown fragment can be determined by running
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standard fragments of known size through the gel in an adjacent well. The gel is stained with
ethidium bromide, a mutagenic chemical that binds to DNA, to visualize the fragments. Finally,
the migration distance of each fragment is compared to the molecular weight standards, and sizes
deduced. You will use this technique to simply visualize your isolated DNA in this exercise, and
to determine several fragment sizes and orientations in the next exercise.
Procedures
Preparation of Suspension Culture (The TA will complete this portion the day before)
- you will do this yourself in later exercises
Sterile technique is required for this protocol. This is important to prevent the growth of foreign
organisms; these will not contain the plasmid you are interested in isolating. Remember, to use a
flame, cover agar plates, and restart contaminated specimens. Be careful not to contaminate agar
plates with good colonies on them, as they will be limited.
1. Use a sterilized culture loop to pick a single colony from your plate of transformed E. coli.
Dig in the agar around the colony. Manipulate the culture loop so that the isolated agar piece
and the desired colony come free from the dish.
2. Inoculate the tube of LB/ampicillin medium (5 ml) by dropping in the agar cube that contains
the bacterial colony. Cutting the agar out this way assures that you obtain a sufficient
amount of cells to culture overnight.
3. Allow the culture to grow overnight in a 37°C shaker.
Plasmid Miniprep – Qiagen Spin Miniprep Plasmid Isolation Kit
(procedure taken from kit instruction manual)
Note: Sterile technique is not necessary for the rest of this exercise. (Why not?) Be sure to
properly label all tubes so that they will not become confused with those of another group.
4. Transfer 1.5 ml of the overnight culture to a 1.5 ml microcentrifuge tube. Balance with
another tube of water (or another group’s sample) for centrifugation. Centrifuge the tube for
5 minutes at 10,000 RPM in the microcentrifuge.
5. Discard the supernatant, transfer another 1.5 ml of overnight culture to the centrifuge tube,
and centrifuge for 5 minutes (you will pellet a total of 3 ml of culture).
6. Discard the supernatant and re-suspend the pelleted cells in 250 l of buffer P1, the cell
resuspension solution. (RNase A has already been added to this solution.) Use the vortexer
at maximum speed to completely re-suspend the pellet.
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7. Add 250 l of buffer P2, the cell lysis solution and gently mix by inverting the tube 4-6
times. The solution should become viscous and slightly clear. DO NOT VORTEX the
solution to mix as this can cause shearing of the DNA. Do not allow this reaction to proceed
for more than 5 minutes or the plasmid DNA will begin to degrade.
8. Add 350 l of buffer N3, the neutralization solution, and invert the tube immediately but
gently 4-6 times to mix. Be sure that the solution is mixed thoroughly in order to avoid
localized precipitation. (It should now be cloudy.)
9. Centrifuge in the microfuge at 13,000 for 10 minutes. Be sure to balance with water or
another sample. While the sample is being centrifuged, prepare one QIAprep spin column.
10. After centrifugation of cell lysate, pipet the supernatant above the white precipitate and add
to the spin column.
11. Centrifuge the column for 60 seconds. Discard the flow-through.
12. Wash the QIAprep spin column by adding 0.5 ml of buffer PB and centrifugeing for 60
seconds.
13. Wash the QIAprep spin column by adding 0.75 ml of buffer PE and centrifuging for 60
seconds.
14. Discard the flow-through and centrifuge for an additional 60 seconds to remove the residual
wash buffer. This is very important! The column wash solution is mostly ethanol. Trace
amounts of ethanol can inhibit reactions that you will be conducting in the following
exercise.
15. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute the plasmid
DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAprep spin
column, let stand for at least 1 min, and centrifuge for 1 min.
16. The DNA is now in solution in the bottom of the tube. Discard the spin column. Close the
tube and set it aside for the next section.
Preparing a 1.4 % Agarose Gel
(NOTE: The gel will be prepared for you for this exercise; however, you will need to
prepare your own gel in Exercise 4, so you should familiarize yourself with this procedure.)
8. Mix up 700 ml total 1X TAE from a 50X TAE stock and Nanopure water. Prior to lab,
calculate the volumes of stock and water.
9. Determine the amount of agarose you need to add to 50 ml of the 1X TAE buffer to obtain a
1.4 % solution (% means grams per 100 ml; a 1% solution would have 1 gram solute per 100
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ml total solution). Mix the solution in a 250 ml Erlenmeyer flask; the agarose won't dissolve
until the mixture is heated.
10. Stuff a Kimwipe loosely into the mouth of the flask. Heat the solution in the microwave for
about 40 to 60 seconds. Try not to let it boil too hard. The Kimwipe is to prevent boiling
over. Careful taking your flask out -- it's hot! Swirl the agarose solution around until you
can't see any more beads of dissolving agarose.
11. Add 1 drop of ethidium bromide (EtBr) for staining. EtBr is a powerful mutagen and a
known carcinogen! Wear double gloves, a lab coat, and glasses. Keep EtBr and all
equipment it contacts in the designated counter space.
12. Allow the agarose to cool until it is comfortably warm (you can hold it against your wrist for
a long time without experiencing pain). Gels poured too hot will warp the gel tray.
13. While allowing the agarose to cool, use laboratory tape to seal the edges of the gel tray.
Arrange the tray in the gel box for pouring the gel. The proper arrangement will be
demonstrated.
14. Pour the agarose slowly into the center of the gel box and allow it to spread to the edges.
Pour the entire volume you mixed up. The resulting gel layer should be about 3mm thick.
Orient the comb on one end of the plate so it rests ~1mm off the plate surface. Check that
there are no bubbles around the comb or in the middle of the gel. When you're happy with
the gel, leave it alone to harden.
Preparing Electrophoresis Samples
15. You will run two lanes of a DNA molecular weight standard and one lane of your isolated
plasmid DNA on the gel. The molecular weight standard is bacteriophage lambda DNA
which has been digested with a restriction endonuclease. (These will be discussed further in
the next exercise.) Obtain two 0.5 ml microfuge tubes for preparing your samples.
16. Prepare a table which lists the lane number, name, and components of each sample that will
be run on the gel (see below for components). The standards should be run on either side of
the plasmid DNA sample.
17. A loading buffer must be added to the samples so that the DNA solution is denser than the
electrophoresis buffer and will sink to the bottom of the gel well. The buffer also contains
two dyes which migrate through the gel with different mobilities so that the approximate
progress of the gel can be measured without actually visualizing the DNA. (The relative
mobilities of the dyes are given in the Equipment and Reagents list.) The buffer is 6X
concentrated; hence, you will add 5 l loading buffer to a 30 l total sample. Use 10 l of
your plasmid DNA and 5 l for each of the two molecular weight standard lanes. Determine
the amount of water you need to add to each sample to obtain 25 l total volume (not
including the loading buffer), and record the amount of each component you will use in your
table. Now, prepare your samples according to this table.
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Loading the Gel
18. Once the gel has hardened (~35 minutes), gently rock the comb back and forth until free.
Strip the tape from the wall of the mold and carefully place the gel in the horizontal
apparatus.
19. Turn your gel around so the running direction is towards the positive (red) electrode. Why
will the DNA run towards the positive electrode? Pour enough TAE electrophoresis buffer
over your gel, being careful to get rid of trapped bubbles, so the buffer comes to about 1 mm
above your gel (you can see this from the side of the gel box). You are now ready to load.
20. The technique for loading samples into a gel will be demonstrated. Draw the sample into the
pipette tip. Position yourself so that you are stable and facing the gel box. Place elbows on
the work surface and place your non-dominant hand against the shaft of the Pipetman to
steady yourself. Lower the end of the tip into the well. Be careful not the puncture the
bottom of the well and not to tear the sides. If the tip is not placed deep enough into the well,
the sample will spill into the buffer. Slowly inject the sample. If the well has been loaded
correctly it will look like a sharp blue square when viewed from the side. Do not take too
long loading the gel, as this will allow the samples to disperse.
21. Check your gel once more for orientation (running direction). Plug the electrode leads into
the power supply. Turn it on. Run for about 30 minutes to 1 hour at a constant 95 volts.
Check the progress of the faint purple band to determine when to stop the gel. The gel
should be stopped when the purple band is 1/2 of the way down. The current will gradually
heat the buffer, and buffer overheating will warp the gel!!! Monitor the gel temperature, and
consult the GSI if it begins to feel too warm (you may need to turn down the voltage at this
stage).
Viewing the Gel
22. Turn off power supply, remove the top of the box, remove the gel tray.
23. Viewing and photographing the stained gel will be demonstrated. Place plastic wrap on the
surface of the UV transilluminator, and press it so that it is smooth. Using the scoop, remove
the gel from the staining solution and slide it onto the transilluminator. Be careful not to
touch EtBr to the non-covered portions of the light table and counter. Turn the light on.
Make sure to wear safety glasses, as UV light will damage your eyes. Do not look at the
gel for more than a few seconds, or you will get “sunburned” by high intensity,
carcinogenic UV radiation. Check you gel through the protective glass. DNA will appear
as pink bands under UV light. Put the Polaroid camera over the table, take a picture of your
gel, and develop it as per the instruction. This will also be demonstrated. Now, DNA will
appear as bright bands against a dark gray background.
Guidelines for Analysis & Conclusions Section
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(Remember, these are points you should consider and include in your analysis. This section,
however, need not be limited to these specific guidelines.)
1. A traditionally used method for the isolation of plasmid DNA from bacterial cells (one that
was commonly used prior to the availability of kits like the one we used) involves cell lysis
followed by centrifugation of insoluble precipitates, just as it occurred in this exercise.
However, the plasmid DNA is then isolated from the supernatant with a series of organic
solvent extractions (e.g. phenol:chloroform) followed by precipitation with ethanol at low
temperatures, and concluding with centrifugation at low temperature to obtain a pellet of
nucleic acids that includes both DNA and RNA. The pellet is then dissolved in a low salt
buffer that contains RNase A. Discuss the advantages of using the QIAGEN kit over this
more traditional method.
2. The isolation method described above is easily scalable. That is, instead of harvesting 1.5 ml
of cells, one could harvest 15 ml of cells and simply increase the amount of reagents used in
each step by a factor of ten. This allows the isolation of a very large quantity of DNA. Does
the QIAGEN kit appear to be easily amenable to scale-up? What would be the limiting
factor for isolating DNA from a large volume of cells using the QIAGEN kit?
3. The lysis buffer (Buffer P2) contains a high concentration of sodium hydroxide (NaOH) and
a detergent called SDS. What properties of SDS (sodium dodecyl sulfate) make it especially
useful for use in a cell lysing solution? What is the mechanism of alkali cell lysis? Be
specific.
4. At what point in the procedure is the chromosomal DNA denatured and how, chemically?
5. The DNA bands on an agarose gel migrate on a log scale, i.e. plotting the log of the
molecular weight versus the distance traveled yields a linear relationship. Hence, the
molecular weight standards can be used to make a calibration curve which can then be
employed to determine the size of an unknown fragment. The sizes of the fragments in the
standard are 12,000 base pairs (bp) through 2,000 bp in 1,000 bp increments, plus 1,650 bp,
1,000 bp, 500 bp, 400 bp, 300 bp, and below. (The low MW bands may not be clearly
visible). Use this information to make a calibration curve and determine the apparent size of
your isolated pUC19 plasmid DNA. Express the value you calculate in kilobase pairs (e.g.
9416 bp = 9.4 kbp), since your gel will not yield more accurate results than that.
6. Agarose gel electrophoresis is used to separate biological macromolecules based on
differences in molecular weight, i.e. lower molecular weight components have a greater
diffusivity through the gel matrix than heavier ones. The plasmid obtained from this
experiment migrates much farther through the gel than one would expect, considering its
molecular weight (pUC19 is 2686 bps). Prior to electrophoresis, the plasmid can be treated
with an enzyme that cleaves the DNA backbone; this results in a slower-moving band.
Explain.
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Equipment and Reagents
LB (Luria-Bertani) / Ampicillin Medium
1 liter deionized water
10 g bacto-tryptone
5 g bacto-yeast extract
10 g NaCl
pH adjusted to 7.0 with 5N NaOH; sterilized by autoclaving.
100 g/ml ampicillin
QiaSpin Miniprep Kit
Sterile culture tubes
Microfuge
Sterile inoculating loops
1.5 ml microfuge tubes
0.5 ml microfuge tubes
Sterile pipet tips and Pipetman
Microcentrifuge tube rack
Electrophoresis unit; includes power supply, box, plate, and sample comb
UV light box
Molecular Weight Marker, Life Technologies (Gibco) 1 kb Ladder
Ethidium Bromide (EtBr), Stock Solution at 10 mg/ml (20,000X)
Agarose, electrophoresis-grade
DNA loading buffer for gels 10X
For 10 mL:
4 g sucrose
0.1 mL 10 mg/mL bromophenol blue
0.1 mL 10 mg/mL xylene cyanol
2 mL 0.5 M EDTA
filter sterilize through a 0.2 m filter
50X TAE Gel Electrophoresis Buffer:
2 M TRIS
242 g/L
0.95 M Acetic Acid
57 g/L
0.05 M EDTA (as sodium salt, pH 8.0) 20.8 g/L
References
Bauer, W.R., Crick, F.H.C., and White, J.H., Supercoiled DNA, Sci. Am. 243(1): 118-133
(1980). [The topology of supercoiling.]
Birnboim, H.C., and Doly, J. (1979) Nucl. Acids Res. 7: 1513-1522.
Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, Part III, pp. 1265-1290,
W.H. Freeman and Company, San Francisco.
Voet, D., Voet, J.G. (1990) Biochemistry, pp. 791-824, John Wiley and Sons, New York.
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Sambrook, Fritsch, and Maniatis. (1989) Molecular Cloning - A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press.
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