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Lab Day 4:
Part I – Gel Electrophoresis
Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Every
molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and
analysis of DNA. We will use agarose gel electrophoresis to verify restriction enzyme digestion of the
pET3a vector.
Gel electrophoresis is a method of separating nucleic acids or proteins based on the rate of movement
while under the influence of an electric field. In the case of separating DNA, gels made of agarose are a
simple to make and economically favorable (as opposed to gels made of polyacrylamide). Agarose is a
polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer
solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to
cool. The result is a flexible gelatin-like slab. During electrophoresis, the agarose gel is submerged in a
chamber containing a buffer solution (TBE or TAE) and a positive and negative electrodes. Sample is
loaded into a well in the gel. An electrical current is then applied across the gel forcing the DNA through
pores within the gel. Under the gel electrical field, DNA will move to the positive electrode (red) and
away from the negative electrode (black). Several factors influence how fast the DNA moves, including;
the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of
the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules.
DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds
to DNA.
Instructions:
Preparing the agarose gel
1. Make 40 ml of a 1.2% agarose gel using 1X TAE. Use an Erlenmeyer flask for the mixture.
How many grams of agarose will you need?
2.
3.
4.
5.
6.
What volume would you use if you had a 50X TAE stock solution?
Melt the agarose in a microwave by microwaving for 30 seconds, followed by several short
intervals - do not let the solution boil for long periods as it may boil out of the flask. After each
microwave session, observe the mixture to make certain that all of the agarose is completely
dissolved. BE CAREFUL: THE AGAROSE WILL BE VERY HOT! Alternatively one can
place the flask into a hot water bath until the agarose dissolves and the solution becomes clear.
Let the solution cool to about 50 – 55 °C, swirling the flask occasionally to cool evenly.
Seal the ends of the casting tray with the provided rubber stoppers.
Place a comb in the gel casting tray.
There are several combs provided, which one will you use?
Into which comp slots will you place the comb?
Once the melted agarose gel has cooled to 50 – 55 °C, add the DNA stain. Ethidium bromide is
commonly used to stain DNA in gel electrophoresis. However, this chemical is a known
mutagen. Thus there is a growing trend to use DNA stains that are safer. In this lab we will use
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SYBR® Safe DNA stain. SYBR® Safe DNA stain is a safer alternative to ethidium bromide (4
– 5 times less mutagenic). SYBR® Safe is provided in a 10,000X concentrated stock solution
and it can be added directly to the melted agarose gel once cooled as stated above.
7.
8.
9.
10.
11.
What volume of the stock solution will you add to the 40 ml of melted agarose gel? Check
your calculation with the instructor.
Pour the melted agarose solution into the casting tray and let cool until it solidifies (it should
appear murky).
Carefully pull out the comb out.
Place the gel in the electrophoresis chamber in the correct orientation.
Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.
Carefully pipette buffer in and out of the wells to remove any potential gel debris. Careful not to
introduce any bubbles, bubbles are bad!
Loading the gel
1. Add Sample Loading Buffer to each 30 l restriction enzyme reaction. The Sample loading
buffer is 6 X. What volume of sample loading buffer do you need to add to the 30 l
restriction enzyme reaction?
2. Record the order each sample will be loaded on the gel and the DNA ladder.
3. Carefully pipette 20 l of each sample mixture into separate wells in the gel.
4. Pipette 10 l of the 1 Kb DNA Ladder standard into at least one well of each row on the gel.
5. Record the manufacturer name and lot number of the 1 Kb DNA ladder.
6. Load the wells in the following order:
Lane
1
2
3
4
5
Sample
1 Kb Ladder
Negative control – no enzyme
Positive control 1 – BamHI only
Positive control 2 – EcoRI only
Double Digest BamHI/EcoRI
Running the gel
1. Place the lid on the gel box, connecting the electrodes.
2. Connect the electrode wires to the power supply, making sure the positive (red) and negative
(black) are correctly connected. (Remember – “Run to Red”)
3. Turn on the power supply to about 90 volts. Maximum allowed voltage will vary depending on
the size of the electrophoresis chamber – it should not exceed 5 volts/cm between electrodes!
4. Check to make sure that a current is running through the buffer by looking for H2 and O2 bubbles
forming on their respective electrode (in this rare case bubble are good!).
5. Check to make sure that the current is running in the correct direction by observing the movement
of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the
DNA).
6. Let the power run until the blue dye approaches the end of the gel.
7. Turn off the power.
8. Disconnect the wires from the power supply.
9. Remove the lid of the electrophoresis chamber.
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10. Using gloves, carefully remove the tray and gel.
Gel Visualization
1. Using gloves, remove the gel from the casting tray and place into the staining dish.
2. View the gel against in a UV box and record the gel image.
Exercise:
Using your printed picture, draw a standard curve for the molecular weight. I will provide the basepair
values for each molecular weight band. Plot basepair size vs. distance of migration. What was the
distance of migration of the two digested pET3a fragments on your gel? According to your standard
graph, what is the calculated basepair for each? How close are your calculated values to the true basepair
values (percentage)?
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1/7/2016 – Biotech 3
Part II – Gel Extraction
The following modified information for DNA extraction from an agarose gel was obtained from a
handbook for the Qiagen QIAEX II Gel Extraction Kit. Please read over the instructions while your gel is
running and be prepared to perform a gel extraction on your digested pET3a plasmid DNA using this
method.
-----------------------Qiagen QIAEX II Handbook Instructions--------------------The QIAEX II Gel Extraction Kit is designed to extract and purify DNA from any agarose gel in either
TAE (Tris-acetate/EDTA) or TBE (Tris-borate/EDTA) buffer, without phenol extraction or ethanol
precipitation. QIAEX II silica particles have been optimized to enhance recovery of very small and very
large DNA fragments. DNA molecules of 40 bp to 50 kb are adsorbed to QIAEX II particles in the presence
of high salt. Nonnucleic acid impurities such as agarose, proteins, salts, and ethidium bromide are removed
during washing steps. The pure DNA is efficiently eluted in just 20 μl of Tris buffer or water, and is suitable
for most subsequent applications; for example, restriction digestion, labeling, ligation, PCR, sequencing,
and microinjection.
Principle
With the QIAEX II Gel Extraction Kit, extraction and purification of DNA fragments is based on
solubilization of agarose and selective, quantitative adsorption of nucleic acids to the QIAEX II silica
particles in the presence of high salt. Elution of DNA is accomplished with a low-salt solution such as Tris
buffer or water.
Solubilization of agarose without sodium iodide (NaI)
The optimized Buffer QX1 in the QIAEX II Gel Extraction Kit efficiently solubilizes agarose, and does not
contain NaI. Residual NaI may be difficult to remove from DNA samples, and reduces the efficiency of
subsequent enzymatic reactions such as bluntend ligation. The standard QIAEX II Gel Extraction protocol
is used to extract DNA from 0.3–2% standard or low-melt agarose gels in TAE or TBE buffer.
A typical agarose gel slice is solubilized by adding 3 volumes of Buffer QX1 to 1 volume of gel (e.g., 300
μl of BufferQX1 is added to 100 mg gel slice) and incubating at 50°C for 10 minutes. The high
concentration of a chaotropic salt in Buffer QX1 disrupts hydrogen bonding between sugars in the agarose
polymer, allowing solubilization of the gel slice. In addition, the high salt concentration dissociates DNAbinding proteins from the DNA fragments. A ≤ 2% agarose gel slice is normally solubilized within 2–3
minutes with Buffer QX1 at 50°C. The incubation with QIAEX II is extended to 10 minutes to complete
the adsorption of DNA to the QIAEX II particles.
Efficient DNA adsorption to QIAEX II — salt and pH dependence
Adsorption of DNA to glass, silica, or diatomaceous earth in high salt is a well known phenomenon.
Incubation of a DNA solution in a highly electrolytic environment with large anions (e.g., from chaotropic
salts) causes a modification in the structure of water, forcing the DNA to adsorb to the silica particles.
Adsorption of fragments smaller than 100 bp is enhanced by increasing the salt concentration, while
fragments larger than 4 kb are adsorbed at lower salt concentrations. Adsorption of DNA to silica also
depends on pH. Adsorption efficiency is typically 95% if the pH is ≤ 7.5, and is drastically reduced at higher
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pH (Figure 1). If the pH of the binding mixture is >7.5, the optimal pH for DNA binding can be achieved
by adding a small volume of 3 M sodium acetate, pH 5.0.
pH indicator in solubilization and binding Buffer QX1
Buffer QX1 in the QIAEX II Gel Extraction Kit is used for solubilization of agarose gel slices and binding
of DNA to QIAEX II silica particles. Buffer QX1 contains a pH indicator, allowing easy determination of
the optimal pH for DNA binding. DNA adsorption requires a pH ≤ 7.5, and the pH indicator appears yellow
in this range. If the pH is >7.5, which can occur if the agarose gel electrophoresis buffer is frequently used
or incorrectly prepared, the binding mixture turns orange or violet. This means that the pH of the sample
exceeds the buffering capacity of Buffer QX1 and DNA adsorption will be inefficient. In this case, the pH
of the binding mixture can easily be corrected by addition of a small volume of 3 M sodium acetate, pH
5.0, before proceeding with the protocol. The color of the binding mixture allows easy visualization of any
unsolubilized agarose, ensuring complete solubilization and maximum yields. The indicator dye does not
interfere with DNA binding and is completely removed during the cleanup procedure.
Washing
DNA molecules bind to the QIAEX II particles during the adsorption step, and non-nucleic acid impurities
such as agarose, proteins, ethidium bromide, and salts remain in the supernatant. A high salt wash with
Buffer QX1 removes residual agarose, and two washes with ethanol-containing Buffer PE efficiently
remove salt contaminants. All traces of supernatant must be carefully removed at each step to eliminate
impurities and reduce buffer carryover. Washed QIAEX II particles carrying adsorbed DNA are pelleted
and air-dried at room temperature (15–25°C) for 10–15 minutes. Drying the pellet is necessary to remove
all traces of residual ethanol, which may interfere with subsequent enzymatic reactions.
Elution in low-salt solutions
Elution efficiency depends on pH and salt concentration. Contrary to adsorption, elution is best under basic
conditions and low salt concentrations when using QIAEX II. DNA is typically eluted with 20 μl of 10 mM
Tris·Cl, pH 8.5, or with water. The maximum elution efficiency is achieved between pH 7.0 and 8.5.When
using water to elute, make sure that the pH value is within this range. In addition, DNA must be stored at –
20°C when eluted with water, since DNA may degrade in the absence of a buffering agent. The purified
DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit
subsequent enzymatic reactions. Elution efficiency also depends on temperature. DNA fragments smaller
than 4 kb are recovered with 70–95% efficiency when eluted at room temperature (15–25 °C) with a 5-min
incubation. For efficient elution of DNA fragments larger than 4 kb, the incubation temperature should be
increased to 50 °C.
After elution, QIAEX II is pelleted by centrifugation. The supernatant containing pure DNA is easily
removed from theQIAEX II pellet, ready for use in subsequent applications.
------------------------------------------------------------------------------------------------------------------------------Now that you’ve read about the background of gel extraction, you’re ready to actually perform a gel
extraction yourself. Follow the QIAEX II Agarose Gel Extraction Protocol below. We will take DNA
concentration measurements of your purified fragment and compete for the highest DNA concentration!
1. Weigh a microcentrifuge tube and record its mass on the side of the tube (as well as in your lab
notebook!).
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2. Excise the larger pET3a DNA band from the agarose gel with a clean, sharp scalpel. In this lab
you will use razor blades. BE CAREFL not to cut yourself when handling the razor blade. To
perform this procedure you will visualize your gel under UV light and cut out the band of interest
from your gel. Wear your PPE (lab coat and goggles) and minimize your exposure to UV light.
Minimize the size of the gel slice by removing excess agarose. Place your cut out gel inside the
weighed 1.5 ml microfuge tube. Use up to 250 mg of agarose gel.
3. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QX1 to 1 volume of gel for DNA
fragments 100 bp – 4 kb; otherwise, follow the table below. For example, add 300 μl of Buffer
QX1 to each 100 mg of gel.
4. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample according to the
table below and mix. What volume of QIAEX II will you add to your sample?
5. Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing*
every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow.
If the color of the mixture is orange or purple, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The
color should turn to yellow. The incubation should then be continued for an additional 5 min at
least. The adsorption of DNA to QIAEX II particles is only efficient at pH ≤ 7.5. Buffer QX1 now
contains a pH indicator which is yellow at pH ≤ 7.5, and orange or violet at higher pH, allowing
easy determination of the optimal pH for DNA binding.
6. Centrifuge the sample for 30 s and carefully remove supernatant with a pipette. CAUTION:
DO NOT PIPETTE OUT THE PELLET! THE PELLET CONTAINS THE RESIN THAT BINDS
YOUR DNA!
7. Wash the pellet with 500 μl of Buffer QX1. Resuspend the pellet in 500 μl of Buffer QX1 and
then vortex.* Centrifuge the sample for 30 s and remove all traces of supernatant with a pipette.
This wash step removes residual agarose contaminants.
8. Wash the pellet twice with 500 μl of Buffer PE. Resuspend the pellet by vortexing*. Centrifuge
the sample for 30 s and carefully remove all traces of supernatant with a pipette. These washing
steps remove residual salt contaminants.
9. Air-dry the pellet for 10–15 min or until the pellet becomes white. If 30 μl of QIAEX II
suspension is used, air-dry the pellet for approximately 30 min. Do not vacuum dry, as this may
cause overdrying. Overdrying the QIAEX II pellet may result in decreased elution efficiency.
10. To elute DNA, add 20 μl of 10 mM Tris·Cl, pH 8.5 or H 2O and resuspend the pellet by
vortexing*. Incubate according to the table below.
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Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH
7.0 and 8.5. When using water for elution, make sure that the pH is within this range, and store
DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can
also be eluted in TE buffer (10 mM Tris·HCl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit
subsequent enzymatic reactions.
11. Centrifuge for 30 s. Carefully pipet the supernatant into a clean tube. The supernatant now
contains the purified DNA.
12. Optional: repeat steps 9 and 10 and combine the eluates. A second elution step will increase
the yield by approximately 10–15%.
Part III – DNA Concentration
Determine the DNA concentration of the eluted sample using the Nanodrop. Record your value in your
lab notebook.
Exercises:
The molecular weight of a dsDNA molecule can be estimated by the following formula:
Molecular weight of dsDNA (Da) = the # of basepairs X 650
1. What is the molecular weight of the fragment that you purified? Hint: Check the pET3a plasmid
map provided in class!
2. Next, calculate the molecular weight of the smaller fragment that was not purified.
3. Calculate the molar concentration of the fragment that you purified. Hint: there’s no hint 