Agarose Gel Electrophoresis

To check PCR products, restriction digests, etc..  Generally use a 0.8% gel.  For separating fragments that are 500bp or smaller, use a 2% gel.  For more detailed information, see the Bio-Rad electrophoresis system  manual.

  1. Pour a 0.8% gel.  Volumes are:

Melt the agarose in 0.5X TBE in the microwave at 20% power until all agarose is dissolved and there are no stringy pieces.  This is about 2 minutes 45 seconds for small short gels. 

Cool the liquid under cold running water for 10-15 seconds or allow to cool by letting it sit at RT until it is not too hot to hold. 

Add a very small amount of ethidium bromide (1ul per 20ml is sufficient).  You can do this simply by dipping the pipet tip into the stock solution and then swirling this into the liquid agar.  Ethidium bromide is used to visualize the DNA when viewed under UV light.  **Ethidium bromide is a carcinogen – WEAR GLOVES WHENEVER YOU ARE NEAR IT AND DO NOT TOUCH ANYTHING EXCEPT ELECTROPHORESIS BENCH AREA WHILE WEARING THESE GLOVES!!!!**

Pour the gel into the gel mold held in place by the clamp, with the desired comb in place.  Immediately rinse the flask in RO water and place on drying rack.  Allow the gel to dry for about 15 to 20 minutes. 

Remove comb and transfer gel in gel mold to gel box with TBE buffer, making sure that the gel is completely submerged (do not fill past max fill line).

  1. Load the gel.

Agarose concentration(%)

Xylene cyanol

Bromophenol blue

0.5-1.5

4-5kb

400-500bp

2.0-3.0

750bp

100bp

4.0-5.0

125bp

25bp

      As the gel runs, you will see two bands separating.  These are the xylene cyanol and the bromophenol blue bands.  Knowing the size of the DNA fragment you are expecting, you can use this to ensure that you have run it long enough. 

      Small-tooth comb wells can hold about 15-20ul

                                               i.     We have 2 ladders.  Sty I ladder is for larger fragments; Hinf I is for smaller fragments.  There are diagrams of the bands near the gel doc system for reference.

                                             ii.     Load 10ul of appropriate ladder every time you run a gel.  You can do one lane or multiple lanes throughout.  Gels without a ladder are worthless if you need to know the size of your DNA fragment.

      After all samples and ladder(s) have been loaded, place the lid on the gel box, make sure it is plugged into the powersource, and turn the powersource on.  Run it at about 92 volts for 30-45 minutes, or until loading buffer bands are where you want them.  If you want to run it slower, turn the voltage down.  Your fragments will run off if you run it too long.  Use the loading buffer bands as a marker!

  1.  Check the gel when it is done using the GeneFlash gel doc system (see GeneFlash protocol).  If its not spread out enough, run longer.  Remember to turn off the power when you disconnect the lid from the box!  Also, when not in use, please make sure the lid is on to minimize evaporation of buffer and to keep dust out.  When you have taken a picture or saved the image, throw the gel away in the trash.

 

  1. Safety:
    1. ALWAYS WEAR GLOVES!!!  Ethidium bromide is a carcinogen.
    2. Dispose of tips used for ethidium bromide in designated waste container. 
    3. If you spill ethidium bromide, cut out the contaminated area and throw away in waste container, along with any other contaminated material (gloves, paper towels, etc.).