Gel Electrophoresis of DNA and RNA

A method for determining the location of a protein binding site, DNase I Footprinting Analysis involves endonuclease treatment of an end labeled DNA fragment bound to a protein. Limited digestion yields fragments terminating everywhere except in the footprint region, which is protected from digestion. DNase I Footprinting Procedure Preparing the DNA substrate: DNA to be…

Electrophoretic analysis of RNA presents unique challenges. RNA is isolated in single-stranded form, without complementary sequences. It must be fully denatured in order to obtain fractionation based on size. However, RNA molecules form complex and in some cases very stable secondary structures, which are more difficult to denature than DNA. Additionally, RNA is extremely vulnerable…

Standard agarose gels can resolve DNA fragments up to 75 kb. Often, analysis of genomic DNA requires resolution of megabase (mb) fragments. Fragments greater in size than one megabase all run at the same rate on agarose gels, (“limiting mobility”), and are therefore not resolved from each other. This reflects the overall mechanism of sieving…

In many cases, the processing of DNA by enzymes is not impeded by agarose. Such reactions can be run directly in bands excised from low melting point agarose gels. The excised band is melted, mixed with the required buffer and enzyme, and then incubated at the optimal reaction temperature. The gel may solidify during the…

The most popular alternative to glass powder elution for the complete purification of DNA from agarose is electroelution. Because agarose gels are run in a horizontal apparatus, the gel can be manipulated during a pause in the run. This allows variations of electroelution to be performed that are not possible with vertical gels, which are…

Restriction mapping involves the treatment of a DNA fragment with restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites. Restriction endonucleases are enzymes that cleave double-stranded DNA at specific sites, generally 4, 6 or 8 base palindromic sequences. Because, in action,…

A variety of denaturants can be used with agarose. Alkaline gels are most often employed with single-stranded Preparing Alkaline Agarose Gels At high temperatures, alkaline conditions will hydrolyze the Agarose polysaccharide chains. To prepare an alkaline gel, the agarose is first melted in water and cooled to near gelling temperature. Buffer concentrate is then added…

Use the tables below corresponding to the AquaPor agarose of choice to determine the percentage gel to cast. (LE = general-purpose; LM = low melting; ES = extra strength; HR = high resolution). AquaPor LE (all-around performance) Gel % Size Range (bp) 2 200-3,000 1.75 250-4,000 1.5 300-5,000 1 400-12,000 0.75 1,000-23,000 AquaPor LM (low…

Agarose electrophoresis is used for a variety of purposes. Specialty grades of agarose have been developed to fulfill specific requirements. The most commonly used variant is low-melting agarose, which has been modified to lower its melting temperature from over 90°C to around 65°C. This allows bands to be excised from a gel and then melted…

DNA and RNA strands are extremely large macromolecules. A 1 kilobase piece of single-stranded DNA or RNA has a molecular weight of 330,000 daltons, larger than the vast majority of proteins. Often in the molecular biology laboratory, genomic DNA fragments even as large as 1000 kilobases (1 megabase) must be separated by gel electrophoresis. The…