Laboratory 4i Making Agarose Gels for Separating and Analyzing DNA Fragments Background Agarose gels are typically used to separate and analyze DNA molecules ranging in length from about 500 to 25,000 base pairs (bp). The ability of a gel to separate molecules is called its “resolving power” and is mainly determined by the concentration of agarose in the gel. Most agarose gels have concentrations between 0.6% and 3.0% agarose in buffer. Agarose gels are prepared by dissolving powdered agarose in a certain volume of electrophoresis buffer. The agarose-buffer mixture has to be boiled for the agarose to go into solution. Powdered agarose may be purchased from a chemical supply house. The buffer may be prepared in the lab or purchased premade, usually as a concentrated stock solution (see Figure 4.17). The most common agarose gel electrophoresis buffer is 1X TAE (TRIS, Acetic Acid, and EDTA). Although used for decades, the TRIS molecule is a rather poor conductor of electricity. Advances in electrophoresis technology have found some buffering molecules that do a better job of conducting electricity than TRIS. One excellent electrophoresis buffering molecule is lithium borate (LB). Lithium borate conducts electric better and doesn’t generate as much heat during an electrophoresis as TRIS does. The result is that LB buffered gels may be run 6–8X faster than TAE gels. Now, many labs use 1X LB buffer to run faster, better gels. Read more about LB buffers at www.fasterbettermedia.com. In this lab manual, agarose gel labs provide the option of either a TAE-buffered gel or a LB-buffered gel system. The concentration of agarose used in a gel depends on the type of molecules to be analyzed. The longer the molecules in a sample, the less concentrated the gel should be. Too high a concentration may impede the movement of molecules through a gel. The lowest practical working concentration of an agarose gel is about 0.6%, or 0.6 g, of agarose dissolved in 100 mL of buffer. This is used when a sample is composed mostly of long DNA fragments. As the concentration of agarose in a gel increases, the agarose threads are pushed closer together, making it difficult for larger molecules to move through them. A higher concentration gel separates short DNA fragments well. Gels of 0.8% and 1% are typical for plasmid analysis. Gels of higher concentration, up to about 3%, may be used with smaller DNA pieces. A lower concentration gel resolves long DNA fragments, such as genomic DNA, best. Purpose To prepare and pour an agarose gel for DNA fragment analysis. Boil agarose and buffer mixture until dissolved. Make sure to add a comb. Figure 4.17. Pouring agarose gels. 88 Chapter 4 Laboratory Manual agarose dissolved in electrophoresis buffer (either 1X TAE or 1X LB buffer) Cool to 65°C, then pour a 7mm thick gel (30 mL for most gel trays).