Archive for August 2011
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, all of which end in one (or sometimes two) of the four nucleotide bases. These fragments are generally radiolabeled to…
Read MoreUsing 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 of the size is established. Size can be expressed as molecular weight or number of bases. It is important to…
Read MoreRun 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 PAGE gels are generally run with a temperature in the range of 45 – 60°C, which is maintained by running the…
Read MorePreparing 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 gel during pouring. Such bubbles are very hard if not impossible to remove. As oxygen inhibits the polymerization process, even…
Read MoreSample 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 to 95°C (see the Dideoxy Sequencing (Taq Polymerase) Protocol for a formula for the loading buffer). Loading the proper amount…
Read MoreDenaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
The denaturation of DNA by urea. 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 RNA strands and remove the influence of shape on their mobility. Nucleic acids form…
Read MoreHorizontal and Vertical Gel Systems – Vertical Tube Gels
Tube gels were used frequently in the development of gel electrophoresis. Although they are still used for some applications (most notably for isoelectric focusing as part of 2D electrophoresis), tube gels have been superseded by slab gels for most applications. Tube gels are cast (as the name implies) in glass tubes of 1-3mm diameter. The…
Read MoreHorizontal and Vertical Gel Systems – The Vertical Slab Gel System
The vertical gel electrophoresis apparatus. The gel is clamped into the apparatus so that the lower end is immersed in the lower buffer chamber, and the upper end forms one wall of the upper chamber. The gel provides the only electrical connection between the two buffer chambers. Cooling is provided by a metal heat sink…
Read MoreHorizontal and Vertical Gel Systems – The Horizontal Gel System
A gel electrophoresis apparatus must allow the researcher to maintain a uniform electric field across the gel, provide cooling to prevent thermal artifacts, and allow access to the gel for sample loading and monitoring the run. Two types of apparatus are in common use: vertical and horizontal. Vertical gel systems are further subdivided into slab…
Read MoreBuffer Additives-Reducing Agents
Disulfide bonds between or within sample protein molecules can lead to the formation of aggregates as well as play a role in the binding of the subunits of many proteins. It is usually desirable to cleave disulfide linkages prior to the protein electrophoresis. For this reason, disulfide bond reducing agents, such as 2-mercaptoethanol or dithiothreitol,…
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