Electrophoresis in practice

As indicated above, electrophoresis represents an important method utilizing an electrical field to move charged particles or molecules with different physical properties through a medium. This section will focus on the set up and use of the basic equipment for analytical gel electrophoresis, which may serve as a precursor (preparative technique) to additional analytical methods including mass spectrometry, PCR and chromatography. 

Materials For this procedure a number of key items are required, which are listed below. It is important to note that, as with many molecular biology techniques, exposiue to certain reagents involved in this procedure (such as ethidium bromide) is dangerous, so appropriate personal protective equipment (particularly gloves) is essential. 

Preparation of sample and buffers DNA is prepared from the acquired sample using conventional DNA extraction techniques and is mixed with a loading buffer containing bromophenol blue, ready to be loaded into the wells on the agarose gel. Typical buffers used for agarose gel electrophoresis are trisacetate EDTA (TAE) or trisborate EDTA (TBE) used to prepare and run the gel. 

Further DNA analysis It is also possible to conduct further steps beyond conventional electrophoresis to allow more advanced analyses of the separated DNA bands. Of these steps, blotting is arguably the most versatile tool for further analysis. As outlined earlier, there are various types of blotting, but the most popular 

3 A labelled probe is then incubated with the membrane 

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Westermeier, R., Electrophoresis in Practice a Guide to Methods and Applications ofDNA and Protein Separations, 3rd ed., Wiley-VCH Press, Weinheim, 2001. 

Westermeier R (1997) Electrophoresis in practice guide to methods and applications of DNA and protein separations. 2nd ed., VCH, Weinheim... 

With V— 102-5 x 104 and z = 1-10, as above, we see that from 10 to 800 distinct peaks can theoretically be resolved in electrophoresis. (In practice the number of resolvable components is much less than nc due to statistical peak overlap as explained in Section 6.7). This enormous resolving power is consistent with observation (see Figures 8.2 and 8.4). Theory thus provides an explanation of the unusual power of electrophoresis and an insight into the variables (z and V) that must be manipulated for increased performance. 

Refs. [i] Westermeier R (2001) Electrophoresis in practice. Wiley-VCH, Weinheim [ii] Townshend A (ed) (1995) Encyclopedia of analytical science. Academic Press, New York, 1068... 

Westermeier, R. (2001) Electrophoresis in Practice 3rd Edition. Wiley-VCH, Weinheim. 

R. Westheimer, N. Barnes, Gronau-Czybalka, and C. Habeck, Electrophoresis in Practice A Guide to Methods and Applications of DNA and Protein Separations, 2nd ed., John Wiley Sons, New York, 1997. 

Westermeier R. Electrophoresis in practice. Part A Fundamentals and proteins. 2nd. edition. New York VCH Publishers, 1996. 

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