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.
- Pour a 0.8% gel. Volumes are:
- Small, short gel = 20ml; 0.16g agarose
- Small, long gel = 30 ml; 0.24g agarose
- Big, short gel = 40ml; 0.32g agarose
- Big, long gel = 60ml; 0.48g agarose
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).
- Load the gel.
- Each sample needs loading buffer, which should be used at a ratio of 1:6. Loading buffer is used as a marker for migration rates and also to give the sample density so that it sinks to the bottom of the well.
- Below is a table taken from the Bio-Rad Electrophoresis manual:
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
- Larger-tooth comb wells can hold about 50ul. This varies with how thick the gel is.
- If larger wells are needed, you can tape two teeth. This is sometimes useful for gel extractions.
- DNA Ladders:
- 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.
- 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!
- Check the gel when it is done using the GeneFlash gel doc system (see GeneFlash protocol). If it's 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.
- ALWAYS WEAR GLOVES!!! Ethidium bromide is a carcinogen.
- Dispose of tips used for ethidium bromide in designated waste container.
- 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.).