How to calculate the length of DNA fragments

Written by simon markham Google
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How to calculate the length of DNA fragments
DNA is the hereditary material in humans and other organisms. (Getty Thinkstock)

DNA research is at the cutting-edge of science but you can get hands-on experience and calculate the length of DNA fragments for yourself if you have access to the right equipment. To do this you first put a DNA sample with restriction enzymes -- these are protein catalysts which split DNA at specific points -- into a special gel. An electric current is then passed through the gel to fractionate the sample. The resulting fragments are then compared to a standard set with a known length -- called a "DNA ladder" -- to size them.

Skill level:
Moderately Challenging

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Things you need

  • DNA electrophoresis equipment
  • Restriction enzyme-treated DNA samples
  • Prepared agarose
  • TBE electrophoresis buffer
  • DNA stain
  • Metric ruler
  • Excel program or logarithmic graph

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  1. 1

    Prepare the gel for the DNA samples. Mix agarose -- which allows different-sized molecules to move through it at different rates -- with enough TBE electrophoresis buffer to fill about a third of a conical flask. The buffer allows electric current to pass through the solution, and the exact amount of agarose needed depends on the size of the DNA samples. Microwave and then mix the mixture until the agarose has totally dissolved, at which point add the DNA stain or dye. When cooled the pour the mixture into the tray of an electrophoresis kit with a comb placed in its base to create moulds. Remove the comb once the gel has set.

  2. 2

    Add the DNA samples to be separated. First add a loading dye to the DNA samples. The dye allows the DNA samples to sink into the agarose gel and helps track how far the sample has gone. Then add enough buffer to cover the surface of the gel. Turn on the current on the electrophoresis machine. Check that bubbles are appearing at the electrodes to verify current is passing through before turning the power off. Next load the DNA samples and a DNA ladder into the gel, put the lid on, and turn the power back on until the dye and fragments have travelled through the gel. Turn off the power and take off the gel box lid.

  3. 3

    Remove the gel from the gel box. When exposed to ultraviolet light the separated bands of DNA should be clearly defined. Take a photo of the gel so you can analyse the reults of the experiment. Dispose of the gel and running buffer.

  4. 4

    Compare the DNA ladder with the bands of the DNA sample to calculate the length of the fragments. DNA length is measured in base pairs (bp). Measure how far the DNA ladder fragments travelled in centimetres and plot the data points on a graph to show distance travelled versus fragment size. The left axis of your graph should show fragment size -- for example 0bp to 1600bp -- and the bottom axis centimetres -- for example 0 to 10 cm. Draw a line of best fit through the data points on your graph to create a standard curve. Simply measure how far the unknown DNA fragment sample travelled in centimetres and interpolate its size based on the standard curve you created from the DNA ladder.

Tips and warnings

  • If you want to do the experiment at home consider an electrophoresis of dye kit. Dye samples can be used repeatedly unlike DNA ones which are also more expensive.

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