Lab # 4: Plasmid MiniPrep, DNA Quantification, and Agarose Electrophoresis
Short Protocol
PLASMID MINIPREP PURIFICATION
A number of simple and rapid protocols are available for preparing small amounts of
plasmid DNA. This procedure is designed to provide a rapid (<1 hr), small scale
preparation of plasmid which is sufficient for restriction analysis or labelling
hybridization probe. A typical 5 ml culture will provide approximately 20 µg of DNA.
The alkaline lysis method has a number of variations, and the incubation times are fairly
non-critical and can be deviated from several-fold. We will utilize one of the numerous
types of commercial miniprep reagents which are now cost-effective, efficient tools for
plasmid minipreps. The plasmid (pCL1) will be used for restriction digestion analyses.
Several Plasmid Miniprepation methods have been established such as the simple and
quick boiling miniprep or those that use diatomaceous earth or cellulose. A boiling
miniprep is sufficient if the plasmid will be used only for a restriction digestion. For
sequencing, the DNA has to be very clean and it is recommended to use one of the
commercial available miniprep kits such as the Quiagen miniprep kit or the BioRad
miniprep kit (manual).
Purpose
To provide experience in plasmid preparation from bacterial hosts, DNA quantitation,
and agarose gel electrophoresis.
Equipment
Prepared solutions (Quantum Prep
Plasmid Miniprep Kit, BioRad #7326100)
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cell resuspension solution
cell lysis solution
neutralization solution
wash buffer
sterile water (preheated to 37°C)
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heatblock/waterbath 37°C
microcentrifuges
pipetors 10, 100, 200, 1000
1000, 200, 10 µl pipette tips
1.5 ml sterile microcentrifuge
tubes
2.0 ml sterile microcentrifuge
tubes
spin filter (Quantum Prep
Plasmid Miniprep Kit, BioRad
#732-6100)
To preserve the culture for future use, remove 200 µl bacterial culture and mix well with
200 µl sterile glycerol. Store at -70°C. Then continue with the plasmid prep. All
centrifugation steps are carried out at max. speed.
I. Wash culture:
1.
2.
3.
4.
5.
transfer 1.5 ml culture to a 1. 5ml sterile microcentrifuge tube
spin for 30 sec
remove supernatant medium
add 200 µl cell resuspension solution to pellet
resuspend pellet by vortexing or pipetting
II. Lyse cells:
1. add 250 µl lysis solution to resuspended cells
2. cap tube and mix by inverting (10 times); DON'T let cells sit for longer than 5
min in lysis solution before you continue; solution should become clear
3. add 250 µl neutralization solution
4. cap tube and mix by inverting (10 times)
5. incubate for 10 min; DON'T let cells sit for longer than 10 min in solution before
you continue, precipitate should be formed
III. Isolate plasmid:
1.
2.
3.
4.
5.
6.
7.
8.
spin lysate at max. speed for 5 min in table top centrifuge
place a spin filter into a clean 2.0 ml centrifuge tube
transfer supernatant (which contains DNA) to spin filter; avoid debris
add 200 µl of mixed matrix to spin filter and pipet up and down to mix
spin for 30 sec
discard the filtrate, add 500 µl wash buffer and spin for 30 sec
repeat step III.6
discard filtrate and spin for 2 min; it is very important that the matrix does not
contain any war buffer
9. remove spin filter and place in a sterile 1.5 ml centrifuge tube
10. add 100 µl sterile water (preheated to 37°C); the water should be added so to the
highest portion of the matrix
11. incubate for 1 min
12. spin for 1 min at top speed
13. remove spin filter
IV. Analysis:
measure the OD at 260 and 280 nm and calculate the concentration of the plasmid DNA
stock solution.
LAB . AGAROSE GEL ELECTROPHORESIS OF DNA
Nucleic acids can be conveniently separated on the basis of size and density using
electrophoresis. DNA is a repeating polymer and thus has a constant charge-to-mass
ratio. Hence, its migration in an electric field is proportional to its mass. In gel
electrophoresis, a supporting medium is used to retain the nucleic acid sample. Several
supporting matrices are available, but agarose is the most commonly used due to its
relative ease and safety of preparation. Because DNA is soluble in water, we first add a
loading dye to the sample which will prevent the DNA from floating out of the gel. In
this exercise, you will prepare and run an agarose gel and fractionate plasmid DNA
samples. Several different agarose gel concentrations will be compared.
Purpose
To gain expertise in the preparation of agarose gels for the separation of DNA samples.
Each group will run the same DNA samples, but will use varying agarose concentrations.
To become familiar with photodocumentation of ethidium bromide-stained gels using the
Eagle Eye system.
Reagents and Supplies
Plasmid DNA samples
MW standards (1 Kb ladder; GIBCO-BRL)
10X loading dye
1X TAE buffer
Agarose
Distilled water
10 mg/ml ethidium bromide staining solution
Electrophoresis apparatus and power supply (EmbiTec, GIBCO-BRL, and
Bio-Rad)
MW standards, linear digest control of pBlueScript vector (Stratagene)
Procedures
A. Preparation of the gel
1. Wearing gloves, insert a clean comb into a casting plate and set the
mold on a level bench.
2. Add the correct amount of agarose to a measured quantity of 1X TAE
buffer in a glass bottle. You will need approximately 30 ml per gel. For a
0.8% gel use 0.24 g agarose.
3. Place the cap loosely on the bottle, and heat the bottle in the microwave
until the mixture just boils. Swirl the bottle to suspend all the agarose and
reheat briefly to ensure that all granules have melted. Leave the lid of the
bottle very loose and to not boil over the agarose.
4. Cool to 60_C (so you can hold the bottle comfortably in your hand),
then pour 15-25 ml into the casting tray from the end opposite the comb.
The gel should be 3-5 mm thick.
5. While the agarose is still liquid, remove any air bubbles to the end of
the tray with a pipet tip.
6. After the gel is set (10-20 min) and appears translucent, remove the tape
and place the gel on the platform in the gel box with the comb toward the
black electrode.
7. Fill the box with 1X TAE to a level that just covers the gel. Carefully
remove the comb as to not tear the sample well.
8. Attach the electrical leads to the gel box such that the positive electrode
(Run to red!) is at bottom of the gel.
B. Electrophoresis
1. DNA samples already contain gel mix and are ready to load.
2. Carefully add 8-10 µl of a single DNA sample to a well. Dip the pipet
tip through the surface of the buffer, center it over the well, and gently
depress the pipet plunger slowly. Be careful not to puncture the wells or to
agitate the buffer solution.
3. Attach the red electrode to the bottom of the gel and turn on the power
supply. DNA is negatively charged and will migrate towards the positive
(red) electrode.
4. Set the power to 100 V. Bubbles should slowly appear along the wire
leads in the gel tank and the dye should begin to migrate toward the far
end of the gel.
5. Electrophorese for about 30 min. Good separation will occur when the
xylene cyanol and bromophenol blue bands have divided the gel into 3
equal parts.
6. Turn the voltage to 0, turn off the power, disconnect the leads, and
remove the casting tray.
7. Stain the gel in 50-100 ml distilled water containing 0.5 µg/ml ethidium
bromide (5 µl of a 10 mg/ml stock) for 5-10 minutes. Caution! Ethidium
bromide is a powerful mutagen; wash skin immediately should you contact
any solution. Transfer the gel to distilled water for 5 min to remove excess
ethidium bromide. Seal the container of ethidium bromide staining
solution for subsequent reuse.
8. View the gel on the UV transilluminator. Wear eye/face protection and
never look at the UV light source with the naked eye.
9. Photograph the gel with the Eagle Eye System as demonstrated by the
instructors.
10. Compare the migration distance to the standards, and compare the
migration patterns between groups using different concentrations of
agarose.
11. Determine approximate DNA band sizes based on migration of the
MW standards.
12. To quantitate DNA band, a NIH Image software can be used.
UV Spectrophotometric Analysis of DNA and RNA
The concentration of an RNA or DNA sample can be checked by the use of UV
spectrophotometry. Both RNA and DNA absorb UV light very efficiently making it
possible to detect and quantify either at concentrations as low as 2.5 ng/µl. The
nitrogenous bases in nucleotides have an absorption maximum at about 260 nm. Using a
1-cm light path, the extinction coefficient for nucleotides at this wavelength is 20. Based
on this extinction coefficient, the absorbance at 260 nm in a 1-cm quartz cuvette of a
50µg/ml solution of double stranded DNA or a 40µg/ml solution of single stranded RNA
is equal to 1. You can calculate the concentration of the DNA or RNA in your sample as
follows:
DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260
unit)
RNA concentration (µg/ml) = (OD 260) x (dilution factor) x (40 µg RNA/ml)/(1 OD260
unit)
In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due
mostly to the tryptophan residues. The absorbance of a DNA sample at 280 nm gives an
estimate of the protein contamination of the sample. The ratio of the absorbance at 260
nm/ absorbance at 280 nm is a measure of the purity of a DNA sample; it should be
between 1.65 and 1.85.
Phenol has an absorbance maximum of 270 but the absorbance spectrum overlaps
considerably with that of nucleic acids. If there is phenol contamination in your DNA
sample, the absorbance at 260 nm will be high, giving a false measure of DNA
concentration. These procedures are specific to the Beckman DU 640B
spectrophotometer.
Procedure for using the to measure OD260 and OD280
1. Turn the machine on using the switch in the back of the machine (lower righthand side as you face the machine, turn on the monitor screen and printer. Wait
for the machine to go through its start up routine.
2. Open the lid on the top of the machine. Choose the small cuvette holder and place
it in the holder in front of the light source. The word FRONT should be toward
the front of the machine.
3. Click on the UV light key to turn the UV on. It takes about a minute for the UV
lamp to warm up. Quit the diagnostic screen by clicking on the word QUIT in the
upper right hand corner. The Main Menu will appear Choose Nucleic Acid from
the menu. When the nucleic acids analysis menu appears, make sure that the
measured absorbances are 260 and 280. Do not use the calculation functions.
4. Use lens paper to clean the surfaces of the cuvette. Rinse the cuvette chamber
with 70% ETOH. Be sure to remove all of the ETOH after the wash. Place 100µl
sample of your blank (nH2O, TE, whatever your DNA or RNA sample is
dissolved in) in the cuvette chamber. Place the cuvette in the holder and place the
lid on the holder.
5. Shut the lid of the machine.
6. Click READ BLANK in the bottom left corner of the screen.
7. Prepare a 1:100 dilution of the sample you want to read.
8. After the machine has read the blank, remove the cuvette, remove the blanking
solution from the chamber, rinse and dry the chamber and place your diluted
sample in it.
9. Click on READ SAMPLES in the upper left hand of the screen.
10. After the machine has read your sample, the data will appear on the screen. You
do not need to print this each time you measure a sample. A machine will collect
data for you.
11. Between samples clean the cuvette as described above. You do not need to blank
each time unless your samples are dissolved in different solutions. If you use a
different cuvette, you must run a blank.
12. When you are finished reading samples, remove and clean the cuvette and put it
away. Print your results by clicking on PRINT at the top right of the screen. QUIT
the data screen. This will take you back to the main menu. Turn off the UV. You
can turn off the machine while the Main Menu screen is active. Turn off the
machine the monitor and the printer.
13. Calculate the concentration of your RNA or DNA sample and the OD260/OD280