Agarose gels act as a matrix which separate DNA based on size. The PCR or sequencing products are loaded into wells within the gel, then an electric current is applied to the buffered gel. The negatively charged DNA fragments migrate toward the positive anode and are separated through the matrix based on size. Smaller fragments move faster through the gel and will appear towards the bottom of the gel or leading edge of the dye front.
We routinely use 1.5% agarose gels which can separate
DNA fragments from 0.2-4 Kb in size. By increasing the agarose to
3-4%, you get better resolution of smaller DNA fragments. The gels
are stained with ethidium bromide to see the DNA fragments under UV light.
Ethidium bromide is an intercalating dye (and a powerful mutagen) which
binds to the DNA and fluoresces under UV light.
1. Make a 1.5% agarose gel according to recipe in Chemicals and Reagents.
2. Place the gel running tray into the chamber perpendicular to the running direction, so the gasket seals and pour the molten agarose to about 4 mm high.
3. Insert the appropriate comb(s).
4. Allow gel to set until opaque (approximately 10-20 minutes).
6. Gently loosen the comb and remove by pulling straight up.
7. Orient the gel so that the wells are near the negative (black) electrode.
8. Pour 1X TBE buffer into the gel rig until the agarose gel is covered. If the buffer does not cover the gel, prepare more from 10X TBE stock.
9. Gently squirt the wells using a glass pipet to
remove any loose agarose.
1. Remove PCR samples from freezer and allow to thaw.
2. Use a Nuncú plate or strip of parafilm
and mix the following, one sample per each well:
4. Load 6-10 ul of the PCR sample (with TBE dye) into the wells. Repeat for each PCR sample.
4. Slide the gel rig cover on securely
5. Turn on the power supply and adjust the voltage to about 100V.
6. Monitor the migration of the DNA fragments by making sure the blue loading dye does not run off the gel.
7. Always turn power supply off before handling
Have someone in the lab show you how to use the equipment
Warning: Over-exposure to UV light will result in severe burns to the skin and eyes. Appropriate protection should be used when taking pictures.
The next steps will be performed in the darkroom located on the first floor of AHRB or in Burt Boyers lab. You will need a key from the lab to unlock the outer darkroom door. Be certain there is no one in the inner darkroom before you enter.
Specific instruction on the use of the equipment will be provided, however, an overview includes the use of:
* Midrange UV Transilluminator (302 nm): stained
DNA is visible by short-wave UV radiation.
* Polaroid Camera: successful gels will be photographed for future reference.
Once a photograph has been taken, you may dispose of the agarose gel in the Ethidium Bromide Waste container (one is located in the darkroom, another in the lab fume hood) or cut out the dyed parts and return to your beaker. Turn off the lights as you leave, lock the outer darkroom door, and return the key to the Cook lab.
* Kodak DC120 digital camera with software
* Closeup lenses and ethidium bromide filter
* Gateway GP6-300. Thatís a PC with 6 Gb drive and 300 Mhz
* Epson Stylus Color 800 printer.
The system allows one to take digital images in the dark, in the light, attached to the computer or remotely. Here are the basic directions for taking, editing, and printing a picture, while attached to the computer.
* Make sure all power and communication cables are
*Turn on the printer. It will make noises for several minutes while it warms up. It may be best to leave this printer on rather than turning off every time.
. Turn on the computer by the gray power switch on the front of the Tower.
. Turn on the camera by sliding the panel on the front.
. On the desktop there is an icon for Photoenhancer - Take a picture. Double click this.
. First, go to FILE and click on TWAIN ACQUIRE
. To set the particulars, go to CAMERA FUNCTIONS
. Here, you will want to set flash (on or off), exposure time, quality of picture (compression), self timer, focusing options, etc. There is a lot here to experiment with, but you will probably find that most of the defaults are OK.
. When done, click UPDATE CAMERA, then CLOSE.
. If all is set to go, click TAKE A PICTURE! [It is possible that there is no more room in the camera. If this is the case (there will be a signal on the screen), the current policy is to start deleting pictures from the back, forward, in case someone had to leave before downloading their pictures. ERASE PICTURES is an option in CAMERA FUNCTIONS.
. Once a picture is taken, you will see a small version of it on the screen. At this point, you will want to TRANSFER the image to PhotoEnhancer. As you see, you can do some enhancement or picture turning here, but you can also do it at the next step.
. Once transferred, enhancement can begin. There are many options here. It may be best to schedule yourself some time to try a lot of them, then build yourself a protocol for your particular needs, whether it is EtBr stained gels, autorads, protein gels, or vacation pictures. You can waste a lot of time trying all the options.
. Probably the best thing to do if the original picture is not optimal is to adjust the camera settings and take it again. The enhancer can only do so much. However, by going to ENHANCE, and BY EXAMPLE, you will see several options on the right side of the screen. These are FOCUS, EXPOSURE, BRIGHTNESS/CONTRAST, and COLOR. In the upper right, you can select a part of the picture by placing the blue square over it. For example, you might have a band on a gel that you want to make sure is brought into focus, or at least is not lost by your manipulations. Select a set of options on the right, and then pick one of the nine options in the main part of the screen. A few seconds later that option will be in the middle. There is an undo button.
. When you are done, make sure to turn off the computer and camera.
* Many pictures do not need to be in color and look better in black and white. To change this, go to IMAGE and CHANGE IMAGE DEPTH. SELECT 256 SHADES OF GRAY. Always remember there is the undo button under edit.
* To save ink and make pictures more appealing, you can reverse the colors. This is best done after changing to grayscale. Go to ENHANCE, click ADJUST, and then NEGATE.
* To save, go to FILE. Save options are tiff, bitmap, flashpix and jpeg. If you want to edit this picture in NIH image (a Mac program) or Scion Image (PC ñ on this machine), you need to have it in tiff format. Itís a good idea to save it in that format here.
* To print, make sure the printer is on and connected. Remember, it takes a few minutes to warm up. If you just want a black and white image, select that and another changes under OPTIONS. This printer can make very good prints with high quality paper.