In studies of transcriptional regulation, it is often necessary to determine the structure and/or amount of a given RNA species. Several techniques have been developed making use of the fact that some nucleases will only…

Differential display is a powerful technique for detecting and quantitating changes in gene expression patterns between differently treated cells. Fragments of those genes which are induced or suppressed can be identified and isolated for further…

Automated sequencing systems make use of fluorescent dye labeling, in combination with laser scanning and computerized data acquisition and processing to carry out the electrophoresis of up to 96 sequencing reactions on a single gel,…

Denaturing PAGE gels for DNA sequencing generally employ 6-8 M urea as their denaturant and TBE as their buffer system. They are poured as described in the section on denaturing PAGE of DNA and RNA.…

In Sanger dideoxy terminator sequencing, the sample DNA is used as a template for a DNA polymerase. Four polymerase reactions are carried out involving enzyme, primer, and sample DNA, along with dNTPs. Each reaction also…

There are four chemical cleavage reactions at the core of the Maxam and Gilbert sequencing system. The figure below left shows an example from these reactions, the reaction cleaving specifically at guanine. The other three…

DNA sequences are determined by a two-step process. In the first step the sample DNA is used, either directly or as a template, to generate sets of fragments. Each set contains multiple lengths of DNA,…

Molecular weight determination is the most basic use of denaturing polyacrylamide gel electrophoresis. Samples are run versus standards of known molecular weight, and a calibration curve of relative mobility (or distance migrated) versus the logarithm…

Temperature The most critical parameter in denaturing DNA-PAGE is gel temperature. Highly concentrated urea, 6-7M, is the most commonly used denaturant, but to be fully effective, the temperature must be maintained above 40°C. Denaturing PAGE…

Sequencing gels are poured between two glass plates separated by spacers. The spacers are typically no more than 0.2mm in thickness. The extreme thinness of the gel allows air bubbles to be trapped in the…