The electrophoretic behavior in gels of DNA and RNA is effected by their size and shape. Both RNA and DNA can exist as single and double stranded molecules and the single stranded forms have significant secondary and tertiary structure. For this reason single stranded nucleic acids are best separated as denatured structures. This is most often an issue for ssDNA after sequencing reactions, and RNA which can be denatured by urea, methylmercuric hydroxide, formamide, form aldehyde and a number of other agents.
Agarose is a highly purified naturally occurring polysaccharide and
preparation of agarose gels involves simply heating the powdered agarose in
buffer to dissolve it. It gels upon cooling. Like acrylamide, the pore size of
an agarose gel is inversely dependent on the agarose concentration. The pores
in agarose gels are generally much larger than those in acrylamide gels and
are widely used in separation of nucleic acids. Low molecular weight nucleic
acids and oligonucleotides, however, are usually separated by PAGE, due to
smaller pore size of the gel matrix.
Agarose gels are simply formed by placing the necessary weight of agarose in the required volume of buffer. This is then heated to boiling to dissolve the agarose. A microwave oven is the fastest way to do this. Before casting the gel the agarose solution is cooled down to 50 °C.
There are many different types of agarose available. The best choice for routine DNA electrophoresis is Agarose for DNA electrophoresis (cat. no. 11404). This offers good gel strength and low impurities that might interfere with subsequent procedures. Other qualities like Agarose SERVA for PCR (cat. no. 11383) are made for efficient separation of small PCR generated DNA fragments. It has a high gel strength for better handling and enhanced visibility due to improved clarity of the gel. For easy recovering of DNA fragments after electrophoretic separation a range of low melting agaroses are available.