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.
More and more, the big scientific reagent venders 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: 1. they are more expensive (about 2x the cost of unprepared ladder) 2. 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) 3. less ladders to choose from (most companies sell only a small subset of their DNA ladders as ready-to-load solutions)
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 should dilute the ladder.
Let’s assume we want to dilute the ladder enough, so that 10 ul of prepared ladder will contain the necessary 1 ug of DNA ladder. 10 ul is a much more reasonable volume to load into an agarose gel. We need to dilute our ladder into two things: 1) agarose gel loading dye and 2) TE. Most agarose gel loading dyes are 5x (including the loading dye I use). So 2 ul of the 10 ul of prepared ladder will be loading dye. With 1 ul being the concentrated DNA and another 2 ul being loading dye, we are left with TE for the remaining 7 ul. Some folks dilute their ladders into water. However, TE is buffered to a high pH and contains EDTA. Both of these features help prevent DNA degradation via DNAses, so it’s better to dilute your ladder with TE. Depending on the volume of ladder you want to run in each lane, you can adjust the amounts of TE and loading dye accordingly (e.g. 20 ul prepared ladder = 1 ul concentrated ladder, 4 ul loading dye, 15 ul TE; 7 ul prepared ladder = 1 ul concentrated ladder, 1.4 ul loading dye, 4.6 ul TE; etc…).
You can prepare your ladder fresh every time, but if you know you’ll always want to load the same volume of ladder, it’s much easier to prepare the entire tube of ladder at the same time. For example with our 100 ug of DNA ladder from NEB, we have enough ladder for 100 lanes. At 10 ul per lane, we need dilute this stock ladder with 200 ul of loading dye and 700 ul of TE. I typically do this directly in the tube provided by the supplier, so I can keep the nice label and easily find the ladder in the fridge. I store the ladder in the fridge, as it’s annoying to thaw the ladder each time you use it (lot’s of folks freeze and thaw the ladder every time they use it; I’ve never had a degradation problem from storing my ladder at 4°C).
If you are really the do-it-yourself kinda person, you can make your own ladder. The simplest way to do so is to cut a plasmid with a set of restriction enzymes to create a nice variety of band lengths across the range you’re interested in. It’s not essential that all of the band lengths are even numbers like 100bp, 200bp, etc… It’s just important that sizes are diverse and within the range you’re interested. To get a decent set of bands where the short bands aren’t too light, you’ll probably need to cut at least two plasmids where one plasmid is focused on shorter fragments. I don’t recommend making your own ladder however, because there are many good ladders already that aren’t very expensive. I think time is better spent understanding life.