ELECTROPHORESIS HEADER-2

Gel Electrophoresis of DNA and RNA

Maxam & Gilbert Sequencing

By National Diagnostics | August 15, 2011 | Comments Off on Maxam & Gilbert Sequencing

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…

Manual Sequencing

By National Diagnostics | August 15, 2011 | Comments Off on Manual Sequencing

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,…

Using PAGE to Determine Nucleic Acid Molecular Weight

By National Diagnostics | August 15, 2011 | Comments Off on Using PAGE to Determine Nucleic Acid Molecular Weight

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…

Run Conditions in Denaturing PAGE

By National Diagnostics | August 15, 2011 | Comments Off on Run Conditions in Denaturing PAGE

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…

Preparing Denaturing DNA & RNA Gels

By National Diagnostics | August 15, 2011 | Comments Off on Preparing Denaturing DNA & RNA Gels

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…

Sample Prep for Denaturing PAGE of DNA

By National Diagnostics | August 15, 2011 | Comments Off on Sample Prep for Denaturing PAGE of DNA

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…

Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA

By National Diagnostics | August 15, 2011 | Comments Off on Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA

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…