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What factors affect the separation and visualisation of DNA fragments in gel ele - (Nov/17/2006 )

greatly appreciate the help I receive smile.gif

-CSI Westminster-

Assuming the gel is an agarose gel...

% agarose - generally higher percentage = higher resolution. You have to make sure that the % is not too high if you are separating large DNA fragments however, because they may not migrate well through the smaller pores of a high % gel. There are threads here on what % to use for different size bands. 1% agarose is generally a good starting point and that may range from 0.5% - 4%.

voltage - generally lower voltage = higher resolution. Running gels slowly tends to result in better resolution than gels run quickly. I think that is because slow running allows all the DNA to pass through the pores, whereas quick running kind of forces it all through at once. 120V is a good starting point, but anywhere from 60V to 150V is fine depending on how long you wish to wait and how well you want your bands to be resolved.

comb thickness - thinner combs = higher resolution. People often overlook this one, but the thinner your combs are, the thinner the bands on your gel are because the DNA starts in a much narrower volume. We have thin and thick combs and the thin combs produce much nicer results

amount of DNA loaded - too much DNA = lower resolution. When you run too much DNA at once, it struggles to all get through the pores and creates a kind of tail on the top end of the band. This often occurs when you run too much cut vector at once. Less DNA also results in crisper bands because there is less DNA at that migration length in the gel. The band/gel photo looks more crisper because there is less fluorescence from the ethidium bromide chelating to such a large amount of DNA. Generally 1 ug (if you have that much) is fine for a single lane holding 40 uL, but in general the lesser the better if you're looking for resolution.

ethidium bromide - when you are attempting to visualise smaller bands or have electrophesed your gel for long time you may need to soak your gel in a bath of 10 ug/mL ethidium bromide for 15 minutes. This is because ethidium bromide has a slightly negative charge and migrates towards the anode (+ve electrode, i.e. top of your gel) during electrophoresis. This results in only a small amount of ethidium bromide at the bottom of your gel and poor brightness in the lower bands. Soaking in ethidium bromide even when you have included it in your gel is a good way to increase the intensity of any band in your gel.