Download Gel electrophoresis of restriction digest

Gel Electrophoresis
Reagents/Supplies:
Agarose
10x TBE
Agarose gel box, tray, comb
Digested DNA
Sample buffer (tracking dye or loading dye)
High-low ladder (molecular weight marker)
Ethidium bromide
UV light box
Overview:
Agarose gel electrophoresis is a simple and highly effective method for
separating, identifying, and purifying 0.5 to 25Kb DNA fragments. The protocol can be
divided into three stages: (1) a gel is prepared with an agarose concentration appropriate
for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the
sample wells and the gel is run at a volatage and for a time period that will achieve
optimal separation; and (3) the gel is stained or, if ethidium bromide has been
incorporated into the gel and electrophoresis buffer, visualized directly upon illumination
with UV light.
Gel electrophoresis can be used for a wide range of applications. It can be either
analytical or preparative and can involve fragments with molecular weights ranging from
less than 1000 Daltons to more than 108 Daltons. The use of electrophoresis to separate
nucleic acids is simpler than its application to resolve proteins. Proteins can have either a
positive or negative charge while nucleic acids have a uniformly negative charge. A
variety of important variables affect the migration of nucleic acids on gels. These include
the conformation of the nucleic acid, the pore size of the gel, the voltage gradient applied,
and the salt concentration of the buffer.
Agarose gel concentration plays an important role in electrophoretic separations,
as it determines the size range of DNA molecules that can be adequately resolved. Low
agarose concentrations of 0.3 to 0.5% are used to separate large DNA fragments (20 to
60kb). For most analysis, concentrations of 0.5% to 1.0% is used to separate 0.5 to 30kb
fragments. High agarose concentrations of 1 to 1.5% can resolve small DNA fragments
(0.2 to 0.5kb)
Voltage applied at the ends of an agarose gel generates an electric field. DNA
molecules exposed to this electric field migrate toward the anode (+) due to the
negatively charged phosphates along the DNA backbone. DNA fragments travel through
agarose at a rate that is proportional to the applied voltage. With increasing voltages,
however, large DNA molecules migrate at a rate proportionately faster than small DNA
molecules. Therefore, higher voltages are significantly less effective in resolving large
DNA fragments. For large DNA fragments it is best to run gels at both low agarose
concentrations and low applied voltages. The best resolution of fragments larger than
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2kb is attained by applying no more than 5 volts per centimeter to the gel (the cm value is
the length between the two electrodes, not the length of the gel).
In general, DNA fragments are separated by size. Large fragments of DNA run
more slowly while small DNA fragments run more quickly. The conformation of the
DNA has an impact on DNA migration during electrophoresis. Closed circular, nicked
circular and linear double stranded (duplex) DNA of the same molecular weight migrate
at different rates through agarose gels. In the absence of ethidium bromide. Supercoiling
winds the molecules up, giving them a smaller radius and allowing them to pass more
readily through the gel matrix. Nicked circular DNA that have lost all of their
superhelicity migrate at a slower rate than supercoiled circular DNA or linear double
stranded DNA. Linear double stranded DNA runs true to its molecular weight. Adding
ethidium bromide to the running buffer introduces positive supercoils and nicking
causing a decrease in the mobility of the DNA. The effect of ethidium bromide can be
seen on both closed circular and linear duplex DNA.
The amount of DNA to be loaded is an important factor in gel resolution.
Between 5 and 200 ng of a single DNA fragment can be loaded into a well. 5ng
approaches the minimal amount of an individual DNA fragment that can be detected by
ethidium bromide staining and 200ng approximates the most that can be resolved before
overloading occurs.
The most common means of monitoring the progress of an electrophoretic
separation is by following the migration of tracking dyes (sample buffer, loading dye,
loading buffer) that are incorporated into the loading buffer. Two widely used dyes
displaying different mobility are Bromphenol Blue and Xylene Cyanol. Xylene Cyanol
migrates with DNA fragments around 5kb and Bromphenol Blue migrates with DNA
fragments around 0.5kb. Bromphenol Blue provides visualization of the mobility of the
fastest fragments and is valuable in determining the length of the gel over which the
separation of DNA has occurred. Xylene Cyanol is useful for the monitoring of longer
runs. Both Xylene Cyanol and Bromphenol Blue contain glycerol which increases the
density of the sample for easier loading.
One the gel electrophoresis has been completed ethidium bromide us used for the
direct visualization of DNA in gels. The dye intercalates between the stacked bases on
nucleic acids and fluoresces when illuminated with UV (260 to 360nm) light. This
allows for very small quantities of DNA to be detected. It is important to understand that
longer DNA fragments will contain more ethidium bromide and appear as a darker band
while shorter DNA fragments will contain less ethidium bromide and appear as a lighter
band. In order to see smaller DNA fragments it may be necessary to add more ethidium
bromide or add more DNA to the well. Ethidium bromide is frequently added to the gel
and running buffer prior to electrophoresis. This step eliminates the need to stain the gel
upon the completion of a separation. This also allows the direct visualization of the DNA
throughout the run until the desired separation is achieved. Ethidium bromide causes
nicks in the DNA fragments in response to UV light. High energy short wavelength UV
light causes more nick in DNA than low energy long wavelength UV light. The nicking
of the DNA may have an effect on the migration of the DNA fragments.
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In addition to the samples loaded onto the gel, at least one lane should contain a
series of DNA fragments of known sizes so that the sizes of unknown DNA fragments
can be estimated. A variety of molecular weight markers are available for the
determination of DNA fragments of various sizes.
Troubleshooting
 Poor resolution of DNA fragments
o Low percentage agarose gels should be used to resolve high molecular
weight DNA fragments. High percentage gels ahould be used to resolve
low molecular weight DNA fragments.
 Band smearing
o Trailing and smearing of DNA bands is most frequently observed with
high molecular weight DNA fragments. This is often caused by
overloading the DNA sample or running gels at high voltages. DNA
samples loaded into torn sample wells will also cause extensive smearing.
 Melting of the gel
o Melting of an agarose gel during an electrophoresis is a sign that either the
electrophoresis buffer has been omitted in the preparation of the gel or the
running buffer has become exhausted during the course of the run. For
high voltage electrophoresis over long periods of time, TBE should be
used instead of TAE as it has a greater buffering capacity.
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Procedure:
Pouring agarose gel
1. Make 60-70ml of a 1% agarose in 1x TBE. Use an Erlenmeyer flask for this solution.
2. Use a Kim wipe or a piece of paper towel as a stopper for the flask and heat the
solution in the microwave for ~2 minutes.
3. Allow to cool to ~60°C. (This should take a couple of minutes. Estimate by touch)
4. Pour the solution onto an agarose gel casting plate.
5. Add the comb to the liquid agarose.
6. Allow to set. (~30 minutes)
7. After the agarose gel has solidified place the gel in the electrophoresis chamber and
add enough 1x TBE to cover the gel. Make sure you place the gel in its proper
orientation so that the negatively charged DNA runs toward the anode(+).
8. Carefully remove the comb from the gel.
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9. Take your four completed restriction digests out of the freezer and prepare them
restriction digest samples for loading.
• Add 2 µl of 10X Gel Loading Dye Mix to each of your four digest tubes.
10. Obtain uncut mini prep DNA from the freezer
• How can you tell whether the enzyme has cut or not? The best way is to
include on your gel a comparison lane containing uncut vector, i.e., vector
DNA that has not been treated with a restriction enzyme. Prepare an uncut
control sample for each vector in the following way
Sterile dH2O
10X Gel Loading Dye Mix
Miniprep Plasmid DNA
8 µl
2 µl
10 µl
= 20 µl
12. Load the DNA samples (all are approx. 20 µl) into wells using a P-20 pipetman set at
20 µl. In addition, load one lane of 10 µl of pre-made DNA size standards known as 1Kb
Plus DNA Ladder.
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13. Record this information in your lab notebook.
14. Attach the wire connectors to the gel cover. (Make sure that the colors match)
15. After loading the gel, attach the lid to the gel box and make sure the leads are
connected in the proper places (DNA migrates to the red-colored anode lead) and
apply a voltage of approx. 100 volts
16. Place the cover on the gel and turn on the power supply. DO NOT REMOVE THE
COVER ONCE THE POWER SUPPLY IS TURNED ON!
17. After you have turned on the gel, make sure that bubbles are coming from the wire
electrodes, indicating that there is current in the gel.
18. Run the agarose gel at the appropriate voltage until the tracking dye has run ~2/3 of
the way down the gel.
19. Turn off the power supply once the tracking dye front has run ~2/3 of the way down
the gel.
20. Remove the gel from the chamber and look at the gel under UV light. Photograph
your gel.
 WEAR UV PROTECTION GLASSES FOR THIS STEP!
 Make sure you are aware of the orientation of the photograph.
 Tape a copy of the photo to your lab notebook and label the lanes.
21. Tape a picture of your gel in your lab notebook.
22. Label the gel and record your results.
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Part II
1. Take a photo of the gel to mock up and print out for your notebook and to send
to the WSSP staff by uploading a mocked–up version of the gel to your school’s
Dropbox on the WSSP Sakai site (https://sakai.rutgers.edu/portal).
2. Import the picture into Powerpoint or another graphics program and crop the
image to only include the gel.
3. Import the cropped gel image into the restriction digest template slide in the Geltemplates.ppt file.
4. Adjust the size and position to closely match the marker and lanes labels.
Individual adjustment of the labels may be necessary. Make any changes to the
text fro the names of the gel, students and clones.
5. Compare the uncut and digest lanes of each clone to determine the restriction
digest was successful.
6. Determine the size of each insert based on the size of the restriction digest
fragment and indicate that on the mock-up of the gel
7. Save the file with the following naming convention: School #, RDG, Gel
number, year (e.g. 20RDG1.10 for the first gel from school #20, 20RDG2.10 for
the second gel, etc.)
8. Send a copy of the picture to the WSSP staff by uploading a mocked–up version
of the gel to your school’s Dropbox on the WSSP Sakai site
(https://sakai.rutgers.edu/portal).
9. Enter the results of the analysis of the clones into your school’s Google-Docs
Clone Management Table.
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