Gel electrophoresis

On Tuesday you will be separating out your PCR products according to size by using agarose gel electrophoresis.

This technique relies on the fact that DNA has a net negative charge (think of all those phosphates along the phosphodiester backbone). So if you put DNA in an electric field it will move towards the positive electrode.... in practice we put the DNA into a jelly-like material called agarose, and the DNA moves through this agarose when an electric current is passed through it.

The cool thing is that large DNA fragments will have a hard time getting through the gel, so they will travel slowly. Small DNA fragments, on the other hand, have much less trouble getting through the gel so they move faster. This means that we can use this technique to separate DNA fragments based on their size.......

Here is what happens with agarose gel electrophoresis with a step-by-step description underneath:

image taken from: http://www.all-science-fair-projects.com/science_fair_projects_encyclopedia/Image:Agarose_Gel_Electrophoresis.png

1. This is a picture of the agarose gel, at the top there are 3 holes (these are called 'wells') where your DNA samples can be loaded
2. This picture shows a DNA sample being added into the first well in the agarose
3. And here the second and third wells are being filled with samples
4. Now all of the wells are filled with sample and the electric current is being turned on, the DNA starts moving towards the positive electrode
5. The electric current has been on for a little while and the DNA has already moved a little way through the gel
6. The smaller DNA fragments have moved right to the bottom of the gel, so now the electric field can be turned off and the gel can be analysed...

In the sixth image above, the sample in the first well was actually a DNA ladder - this is a solution containing a mixture of DNA fragments known sizes. These different size DNA fragments will move through the gel at different speeds - the small fragments will move faster and so end up at the bottom of the gel, and the larger fragments will move slower and end up still near the top of the gel.  We use DNA ladders containing DNA fragments of known sizes to estimate the size of the DNA in our samples.

Here is an example of a DNA ladder - notice the smaller fragments (measured in base pairs - bp) are near the bottom of the gel :

image taken from: http://www.diytrade.com/china/pd/5128543/1Kb_DNA_Ladder_Plus.html

That seems like a lot of information for a relatively simple technique! Hope it helps...