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…
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…
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…
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…
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…
Restriction endonucleases are enzymes that cleave double-stranded DNA at specific sites, generally 4, 6 or 8 base palindromic sequences. Because, in action, the enzymes are sequence-specific, each piece of DNA has a recognizable pattern (or…
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…
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…
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…
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…