Agarose Gel Electrophoresis Of DNA
SP, Jul 04
Negatively charged nucleic acids (RNA and DNA) can be conveniently size-separated using gel electrophoresis. For molecules of small size (less than 100-150 bp), acrylamide gels are used (DNA sequencing, primer extension analysis, etc.).
Agarose gels are routinely used to separate DNA molecules of upto >2 Mb size (pulsed field electrophoresis is needed for DNA of more than 25 kb size).
In an electric field, the molecules migrate at a speed that is inversely proportional to the log(10) of their molecular weight.
At high voltage, large molecules move faster than they are supposed to - so high voltage should not be used when separating large DNA fragments. Large gels are run at 1-2 V/cm gel length overnight while minigels are generally run at 10-15 V/cm for an hour or so.
The speed of migration is also affected by the concentration of agarose (0.8-1% is good in most cases).
0.5% - good for 1-30 kb
1.5% - good for 0.2-3 kb
It should be remembered that circular and supercoiled DNA (such as plasmids) migrate faster (more so in the absence of ethidium bromide) than linear DNA of same size. In general,
For double-stranded DNA, circular but nicked < linear < circular but not supercoiled < supercoiled (fastest). For single-stranded DNA, circular < linear
Ethidium bromide staining can detect as low as 5 ng of DNA. Saturation is reached around 200 ng and more DNA than that can cause appearance of leading trails.
The runs are usually done in tris acetate EDTA (TAE) buffer. Tris borate EDTA buffer (TBE) is rarely used in the lab, though it has higher buffering capacity and is better for high voltage runs. TBE, however, is not well suited for (DNA from) gel purification protocols.
TAE buffers can be reused after runs (usually 4-5 times).
The DNA staining dye, ethidium bromide, is added to the buffer at 0.5-1 ug/ml when the gel electrophoresis tanks are filled. It is not necessary to have the dye in the agarose gel when gel casting is done. If run is performed in buffer without the dye, post-run staining can be done in 0.5 ug/ml ethidium bromide in water for 20-30 minutes.
Xylene cyanol and bromophenol blue dyes usually present in loading buffer can prevent good visualization of DNA fragments they comigrate with. In such cases, place the gel after run in water or TAE buffer for 30 minutes or so to allow the dyes to diffuse away.
Agarose of molecular biology grade should be used. Note that it is possible for the agarose and the running buffers to be contaminated with nucleases (will cause cloning problems).
See 'Solutions' section in this wiki for composition of loading buffer and TAE buffer.
1. Weigh out the right amount of agarose (use minimum amount; agarose is expensive; 50 ml for a minigel) and melt it in TAE buffer in a glass bottle (with lightly placed cap; volume of bottle should be ~3x volume of TAE/agarose) in a microwave oven (~2 min for 100-200 ml volume).
2. Use insulated gloves to remove the bottle (holding it around the neck, with its cap loose and its mouth pointing away from you), and place it in a warm water bath (around 50 deg) to let it cool to a temperature which will let the agarose be liquid yet not damage the casting apparatus. You can cool the bottle in a room temperature water bath if in a hurry. In such cases, do not overcool (gel will solidify) and occasionally swirl the bottle (to prevent liquid near the bottle wall from solidifying).
3. Pour the gel in the cast. Make sure that the gel tray and the comb is clean.
4. Allow 15-30 minutes for the gel to solidify (can be hastened by placing it at 4 deg).
5. Fill electrophoresis tank with TAE buffer (~400 ml for a minigel tank). You can reuse old buffer 4-5 times, but use new buffer for good pictures and crucial experiments.
6. Add ethidium bromide to 0.5 ug/ml.
7. Load DNA in loading buffer (15-25 ul volume can be loaded in each well depending on comb size).
8. Run at 80-90 volts for an hour or so.
9. Use an UV imager to take a picture. A handheld UV illuminator or a table top transilluminator can be used too.