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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.

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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 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

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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 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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10. Each student should prepare the samples from their PCR reactions by adding 2.5 µl of 10X Loading dye to each PCR tube. 11. Load the DNA samples (all are approx. 20 ml) into wells using a P-20

pipetman set at 20 ml. In addition, load one lane of 10 ml of pre-made DNA size standards known as 1Kb Plus DNA Ladder.

12. Attach the wire connectors to the gel cover. (Make sure that the colors match) 13. 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

14. Place the cover on the gel and turn on the power supply. DO NOT REMOVE THE COVER ONCE THE POWER SUPPLY IS TURNED ON! 15. 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. 16. Run the agarose gel at the appropriate voltage until the tracking dye has

run more than ½ way down the gel

17. Turn off the power supply once the tracking dye front has run ~2/3 of the way down the gel.

NOTE: The running buffer can be recycled and used multiple times!!! Pour the running buffer back into your 1X TAE roller bottle with a funnel at the sink. We often use our gel buffers 5-10 times. 18. Take a digital photo of the gel and transfer the jpg file to a computer. 19. Tape a picture of your gel in your lab notebook. 20. Label the gel and record your results.

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Interpret your PCR reactions Now that you have taken a photograph of your agarose gel it is time to interpret the data that you obtained. The main thing that you want to learn from your agarose gel electrophoresis is “How big are the inserts in each of my plasmids?” To do this you will first need to mock up your gel, interpret the data, send the mocked up version of the gel to your school’s folder on the Sakai site and enter your conclusions into the Clone Report sheet for you school on Google Docs.

Lab 4: Agarose gel of PCR Samples

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1. Open the picture in Powerpoint or another graphics program and crop the image to only include the gel. Often the image taken by the camera has a lot of extra space around the edges and has labels that are hard to read (Fig 2). To create a better presentation, import the gel image into Powerpoint or a picture editing program to edit the image to only include the desired lanes (Fig 3).

2. Download the PCRG.ppt file posted on the WSSP and Sakai sites. You will use this file as a template for mocking up your gel images to determine the size of the clones. Using the template file will save you time in having to create all the different labels. The template file includes instructions for mocking up the images and template for the PCR gels done in this lab.

3. Import the cropped gel image into the appropriate gel template and size and position the gel to get it to align to the marker and lane labels as close as possible.

Note that this will not be perfect. You will likely have to individually move the markers and labels to properly align with the DNA bands in the gel image.

4. Change the labels to match your gel and clone names. 5. Determine the size of each insert based on the size of the

PCR fragment and indicate that on the mock-up of the gel (Fig. 4).

6. Save the file with the following naming convention: School #, PCRG, Gel number, year (e.g. 20PCRG1.10 for the first gel from school #20; 20PCRG2.10 for the second gel, etc.)

To make the file size smaller, please delete instruction, template and examples slides that are not your gel.

7. Upload the ppt file of the mocked–up version of the gel to your school’s Dropbox on the WSSP Sakai site (https://sakai.rutgers.edu/portal).

Fig 4 A properly mock gel with PCR screen samples. Fragments above the white dashed line have PCR fragments greater than 500 bp, indicating the cDNA inserts are larger than 300 bp.

Fig 2 A gel image before cropping.

Fig 3 A gel image after cropping

Lab 4: Agarose gel of PCR Samples

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i) Log onto the WSSP Sakai site (https://sakai.rutgers.edu/portal) using your school’s username “wssp<school #><school initials>@gmail.com” i.e. wssp20ru@gmail.com for Rutgers University. Ask your teacher for the password for your school’s account.

ii) Connect to the WSSP tab near the top of the page (Fig 5).

iii) Connect to the Drop Box link on the menu bar on the left (Fig 6).

iv) Select the Upload Files in the Add pull down menu of your schools Drop Box folder to upload the file.

Please use the proper naming of the gel files. Your school may run as many as 30 or gels and so it is very important that we are able distinguish between them.

8. Enter the results of the analysis of the clones into your school’s Google-Docs Clone Management Table (Rows 2-9, Fig 7). i) Determine the size of the DNA fragments

that result from each PCR reaction. By comparing the distance that your fragments have migrated relative to the DNA standards (Fig 4), you can determine the approximate size (in nucleotide pairs) of your fragments.

ii) Determine the size of the insert by subtracting the 200 bp of the PCR fragment generated from the vector.

iii) Identify clones with > 300 bp

inserts. Since 200 bp of the PCR fragment is from vector sequence flanking the insert, the size of the PCR fragments will be over 500 bp. In the next lab you will be doing plasmid DNA minipreps on these clones. Clones with smaller than 300 bp inserts will not be prepped.

Fig 7 Google Docs Clone report sheet. The PCR gel name and insert size data for the clones shown in Fig 4 are properly entered into rows 2-9.

Fig 5. Sakai page after logging in. Connect to the WSSP tab.

Fig 6 The Upload Files option is shown in the Add pull down menu of the Drop Box folder.