Preparation of DNA ladder for agarose gels

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Preparation of DNA ladder for agarose gels

Summary: When you first start in a lab, it can be tricky to learn how to appropriately dilute the concentrated DNA ladders provided by experimental biology suppliers into a form suitable for agarose gel electrophoresis. You must dilute the DNA ladder stock with the appropriate amounts of TE and agarose gel loading dye. If you don't want to spend time preparing your DNA ladder, many biosupply companies provide ready-to-load ladders for about 2x the cost of concentrated ladder.

Related IzziD Article:
Simple, cheap loading dye for Agarose gel electrophoresis

Article created: Oct 8, 2007
Article by: Jeremiah Faith

After working at the lab bench for over four years, I feel an article on how to prepare a DNA ladder for agarose gel electrophoresis is not terribly useful to many folks. However, when I started doing experiments as a first year graduate student, I dreaded the thought that the ladder would run out when someone wasn't around to help me prepare more. I had no idea how to prepare the thing, but it was essential to have it so I could estimate the lengths of DNA fragments on my gel. After a couple months, I got a recipe from someone, but I still didn't really understand why I was adding the particular components at their different concentrations (and thinking back, the components in that particular recipe weren't really ideal).

I'm going to describe step-by-step how to prepare DNA ladder for gel electrophoresis in a few different ways. If you're not a lab newbie, this article probably isn't for you.

The simplest route to DNA ladder

More and more, the standard scientific reagent supply companies are providing ladders that are already diluted appropriately and contain loading dye. With these premixed ladders, you need only add the correct volume (usually 10 ul) of premixed ladder into a well in your gel. As long as you use them up in a few months or so, you can keep the ladders in the fridge (4°C). That way you don't have thaw them every time you run a gel. Examples of these ready-to-load ladders are the Fisher ExactGene Ladder (1KB Plus is a good general purpose ladder; Part No: BP2579-100) and the NEB Quick-Load Ladder (2-Log is a good general purpose ladder; Part No: N0469S).

The problems with prepared ladders are:

they are more expensive (about 2x the cost of unprepared ladder)
the load volume is fixed (e.g. if you are running a thin gel that will only hold 5 ul of dye, but you need 10 ul of ladder, you'll be stuck with a faint ladder)
less ladders to choose from (most companies sell only a small subset of their DNA ladders as ready-to-load solutions)

A more common (and more cost-effect) route to DNA ladder

It's probably better in the long run if you learn to prepare the ladder yourself. Unless you run a huge number of gels, you won't have to prepare the ladder too often. DNA ladder is typically packaged as a concentrated DNA solution in TE. To prepare it you need to dilute it appropriately and add agarose gel loading dye. It is sometimes tricky to figure out how much of the concentrated DNA you should run per lane. Most vendors provide a small picture of the ladder on a gel when they send you the product. Typically, the amount of DNA they used for the gel will be written as a caption under the picture. This amount is usually a reliable amount to use per well in your own gels.

Let's do an example. The DNA ladder I use most frequently is the 2-Log ladder from NEB (Part No: N3200S). I like the ladder because it has a large number of bands across a wide range. One thing in particular I like about NEB ladders is that they spike in a few of the bands at an extra high concentration relative to the other bands. This helps you quickly get your bearings when you look at the gel, helps quantify the amount of DNA in your other lanes, and provides at least a few reference points if your gel turns out really faint for some reason. If you can't see those high concentration bands, you've probably made an error like forgetting to stain your gel.

If you order 2-Log ladder (N3200S) from NEB, they send you a 1.5 ml tube with 100 ug of DNA ladder at a concentration of 1000 ug/ml (1 ug/ul). In the picture provided with the ladder they say, "2-Log DNA Ladder visualized by ethidium bromide staining on a 1.0% TBE agarose gel. Mass values are for 1 ug/lane". So, at the stock concentration the ladder is provided in, you would only need to add 1 ul (i.e. 1 ug) of ladder to your gel to have a reasonable looking gel. However, you would at least need to add loading dye to the DNA to prevent the ladder from floating out of the gel after you loaded it. Even with loading dye, you'd only be adding 1.2 ul to your gel, which is too small to be easily loaded into an agarose gel. Therefore, we must dilute the ladder.

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