Double-stranded DNA is not a completely straight rigid rod. Sequence variations can cause bends in the double helix, or even alter the basic structure of the helix. A bend or kink in the DNA restricts…

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…

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…

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,…

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…

PCR reactions produce product in a nonlinear pattern (See figure below). Amplification follows a typical exponential curve until some saturation point is reached. Generally, products will not be further amplified once 1-5 µg has been…

The polymerase chain reaction (PCR) is a powerful technique which uses repetitive cycles of primer annealing, primer extension, and product denaturing to produce an exponential increase in the copy number of the target DNA. Two…

Native PAGE gels are prepared by mixing an acrylamide/bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate, and water to achieve the desired gel concentration. TEMED and ammonium persulfate are added to initiate polymerization and…

Sample preparation for native PAGE is straightforward. Since the DNA does not need to be denatured, one is concerned mainly with buffer content, density, and visibility. The salt content of the sample should be adjusted…

In the absence of denaturants double stranded DNA retains its double helical structure, which gives it a rodlike form as it migrates through a gel (for non-denaturing electrophoresis of single stranded DNA, see SSCP Analysis).…