Preparation of DNA ladder for agarose gels (page 2 of 2)

Search

Topics

Home

Subscribe in a reader

Other IzziDs

Site Map -- Disclaimer

Preparation of DNA ladder for agarose gels

(page 2 of 2)

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...).

It is convenient to prepare the ladder in the tube it comes in.
Click image for larger

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).

The complete do-it-yourself route to DNA ladder

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.

Previous Page

Skip to page: 1 | 2