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…

DNA samples for denaturing gel electrophoresis must be denatured prior to loading, to avoid time dependent denaturation artifacts on the gel. This is usually carried out by diluting the sample into 95% formamide and heating…

The electrophoretic analysis of single-stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents which unfold the DNA or…