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).…

Uracil or methylation interference assay. End labeled probe is modified at one site per molecule, and allowed to bind protein. Bound and unbound populations are separated, and strands are cleaved at the modified bases. Bases…

The binding of proteins to specific DNA sites is an important mechanism of cellular regulation. Numerous techniques have been developed to analyze the interactions of regulatory proteins with DNA. Three techniques are presented here which…

Primer extension is another technique used to analyze RNA structure and expression. In this method, an oligonucleotide primer is annealed to RNA and extended to a cDNA copy by reverse transcriptase in the presence of…

Ribonuclease protection is a procedure that uses uniformly labeled RNA probes to analyze sample RNA. In this case, probes are chosen to fall entirely within the coding region, so they are only digested if no…

Nuclease S1 will digest only ssDNA or ssRNA. If a duplex of DNA and/or RNA strands have single-stranded overhangs or unhybridized internal loops, these will be digested away. The remaining intact nucleic acid fragments represent…