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…

SSCP (Single Strand Conformational Polymorphism) Analysis. Single point mutations can cause major differences in the folded form of single-stranded DNA. These differences can be detected as differences in electrophoretic mobility. Single-stranded DNA can adopt multiple conformations under non-denaturing conditions. In the absence of a complementary strand, DNA will anneal short internal complementary sequences, forming a…

Heteroduplex Analysis. Annealing of mutant DNA to wild-type probe gives duplexes with one or more mismatched bases (heteroduplexes). Mismatching causes the double helix to take on a conformation that retards its mobility during electrophoresis. Double-stranded DNA is not a completely straight rigid rod. Sequence variations can cause bends in the double helix, or even alter…

Native DNA PAGE gels can be used to detect small mutational differences between DNA molecules. In heteroduplex analysis, for double stranded DNA, the basis of separation is the conformational difference arising from the bending of the rodlike double helix caused by small mismatches between the strands. In SSCP analysis, single stranded DNA molecules are fractionated…

Methods of DNA/RNA Purification from PAGE Gels. Polyacrylamide gel electrophoresis yields individual bands of extremely high purity, in that only one nucleic acid sequence tends to be present in each. Electrophoresis can thus serve as a powerful purification tool for DNA or RNA. In practice, most purifications are carried out on DNA, which is more…

Protein bound to a small piece of DNA will alter the electrophoretic mobility of that DNA fragment. This allows the analysis of protein-DNA interactions, including the measurement of binding rates, affinity, and specificity. In addition, bound and unbound DNA may be isolated from the gel and used for further types of analysis such as methylation…

Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and yield. PCR products can range up to 10kb in length, but the majority of amplifications are at 1kb and below, where PAGE analysis is the most effective. In the size range from 400 to 1000 bases, the choice of native PAGE…