IGEM:Caltech/2007/Protocols/DNA Gel Electrophoresis
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(New page: ==Materials== *TAE (1X) *GelStar (DNA stain) *DNA *Nanopure water *loading buffer (6X) *ready-to-load ladder stock *assorted pipetmen *assorted tips *100 ml graduated cylinder *ki...)
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Revision as of 21:25, 19 June 2007
- TAE (1X)
- GelStar (DNA stain)
- Nanopure water
- loading buffer (6X)
- ready-to-load ladder stock
- assorted pipetmen
- assorted tips
- 100 ml graduated cylinder
- kim wipes
- gel box, tray, comb, and power supply
- 0.6 ml microfuge tube
- bench top microfuge
- Gel Doc XR gel imager
- 250 ml Erlenmeyer flask
- weighing dish
- Prepare the gel tray for pouring of the gel solution by placing it into the tray apparatus and tightening the sides of the apparatus into place around the tray. Make sure that the pouring apparatus and tray are level.
- Remove the GelStar stain from the -20C freezer and thaw at room temperature (protected from light) for 10-20 minutes. Once thawed spin the vial quickly (brief pulse) in the benchtop microfuge to deposit the solution to the bottom of the vial.
- Based on the size of the DNA you will be separating on the gel, determine the appropriate percentage gel to run (typical applications require between 0.7% - 1.0%). For smaller fragments (less than 1 kb) you may run a 2.0 – 3.0% gel. Note that these percentages are in weight per volume units.
- Make up the gel solution in a 250 ml Erlenmeyer flask. The total volume required for one gel is 50 ml. Weigh out the appropriate amount of agarose using a metal spatula and weighing dishes. Carefully pour the agarose into the flask. Measure out 50 ml of 1X TAE in a graduated cylinder and pour the buffer into the flask over the agarose powder. Mix by swirling. Note that the agarose will not go into solution.
- Cap the flask loosely with a kim wipe and put it into the microwave. Heat the mixture to boil in the microwave, being carefully not to allow the mixture to overflow. Typically heat on high for 30 seconds, remove and swirl briefly, and then heat for another 15-30 seconds. Swirl the mixture so that the agarose is melted and then cool by either allowing the solution to cool slowly on the bench top or by running it under tap water. Once the mixture has cooled (the gel mixture should be warm to the touch) add 2.5 µl of GelStar to the 50 ml gel solution. Swirl to mix and pour 50 ml of the gel solution into the gel tray. Place the appropriately sized comb into place and make sure that it is situated correctly in the gel. Allow the gel to cool in the tray and solidify.
- While the gel is solidifying (approximately 15-25 minutes), prepare the DNA solutions to be run. For the small combs, solutions of 6 µl are typically run; for the large combs, solutions of 12 µl are run (although these volumes can be varied slightly). In a 6 µl solution add 1 µl 6X loading buffer, x µl DNA, and 5-x µl nanopure water, scaling as appropriate using the L20 pipetman.
- Select the appropriate DNA ladder (either 1 kb or 100 bp DNA ladder; see attached image of fragment sizes) depending on the sizes of the DNA to be separated. The ladder comes in a ready-to-load solution.
- Once the gel has solidified remove it from the pouring apparatus and place it carefully into the gel box. Orient the gel such that the wells are closest to the cathode side of the gel box (black). Pour 1X TAE into the box such that the gel is completely, but just, covered. Make note in your laboratory notebook how the samples will be loaded and load accordingly.
- Samples should be loaded using a L20 pipetman set to the appropriate volume. Mix DNA samples prior to loading by pipeting up and down briefly. When loading a gel, it is easiest if there are no air pockets in the pipet tip. Also, you will most likely have to stabilize your loading arm either on the bench top or with your other arm. You’ll also have to position yourself so that you can see the wells and place the pipet tip just over the well. Do not insert the tip too deeply into the well as you will likely pierce through the well.
- Once loaded run the gel at constant voltage at approximately 12 V/cm (typically between 80-100 V). This setting can be modified depending on whether you want the separation to occur faster or slower; however, we typically do not run gels of this size faster than 100 V. Better resolution will be obtained at slower separation speeds.
- Monitor the movement of the DNA through the gel, by monitoring the location of the loading dyes in the gel as it runs. The DNA will move towards the anode side of the gel box (red). Stop the separation when the faster moving dye (bromophenol blue) is approximately 75-100% across the length of the gel (adjusted as appropriate for the fragments you are separating). Turn off the power supply and carefully transfer the gel onto the light source of the gel imager. The gel does not need to be removed from its tray.
- Image and take a picture of the DNA on the gel (follow the next section to learn how to take a picture using the gel imager)
- The gel can be placed back into the gel box and reused (fresh wells only) if space is available on the same day only.
- If performing a gel extraction use the prep-UV setting (longer wavelength) on the gel imager. Make sure to use the shield while cutting out the bands with a clean razor blade. Dispose of the razor blade into a sharps container.
Additional Method: Using Gel Doc XR and Quantity One software to image gels
- Turn on the Gel Doc XR gel imager. The ON/OFF switch can be found on the lower left rear of the machine. Once the machine is on, the “POWER” indicator on the control panel (front of the machine) will be lit.
- Open up the software program Quantity One on the computer station to the right of the Gel Doc XR. The shortcut can be found on the desktop.
- Open up the gel drawer on the Gel Doc XR and place the gel on the center of it. The bottom of the gel (the edge the DNA was migrating towards) should be placed closest to you. Close the gel drawer.
- On the “Quantity One Basic” shortcut menu display, click the icon left of “1. Select Scanner”. Select “Gel Doc XR …”
- A window will open up showing an image screen.
- Press the “TRANS UV” button on the control panel of the Gel Doc XR. This turns on the UV light.
- The gel window should be running in “Live/Focus” and a preliminary image of the gel should be seen.
- Following the steps outlined on the left side of the gel window, first follow step I “Position Gel”. If necessary, zoom in and out (“Tele” or “Wide”) such that the entire gel is seen. Since the same sized gels will be run in this laboratory, only in rare circumstances will zooming, focusing, and iris opening need to be altered.
- Under “Step II. Image Mode”, make sure that “UV” is selected.
- Under “Step III. Acquire Image”, there are two methods shown. Both are based on exposure time. “Auto Expose” will cycle through several exposure times and find the optimal one. “Manual Acquire” lets you set your own exposure time in seconds. I recommend using the “Auto Expose”.
- Once the image you want is displayed in the window, press the “Freeze” button to stop image acquisition.
- Under “Step IV. Optimize Display”, select “Invert Display.” When taking UV images of DNA gels, you will see a dark background with bright (white) bands. To save printer toner and to simplify labeling, we invert the images such that the background appears light and the bands appear black. When you print the image you may write directly on it for labeling.
- Click on “Analyze”. This will place your image in a separate window.
- Select the image in its new window. You can still optimize the display and invert the image here by right-clicking the image and hitting “Transform…”.
- Once the image is to your satisfaction, go to “File/Save” and save your image in your folder under “My Documents”.
- To print the image, right-click on the image and select “Print Image…”
- Press the “TRANS UV” button again to turn off the UV lamp. Open the gel drawer to remove the gel.