Denaturating Nucleic Acid Electrophoresis

General differences between DNA and RNA in denaturating electrophoresis do not exist therefore, the following protocols maybe used, as well as for DNA and RNA. 

Instead of EtBr use less harmfull fluorescent dyes, e.g., propidium iodide (PI) or SYBR Green (cf. Haugland RP (1996) Handbook of fluorescent probes and research chemicals, 6th ed.. Chap. 8, Molecular Prohes, Eugene, Oregon https //catalog.invitrogen.com) 

If ethidiimi bromide is omitted as well as in gel and electrode buffer, the DNA sample is supplemented with 5 pi of Soln. D per milliliter and incubated for 10 min at RT. 

A gel of 2-8 mm thickness is made from Soln. C in a horizontal slab apparatus. 

The RNA sample is incubated in Soln. B for 1 h at 50 °C. After cooling, the solution is mixed with 1/10 of its volume with Soln. D and loaded into the slots of the gel. 

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Electrophoresis is without doubt the most convenient and accurate method for determining the size of nucleic acids (Sect. 4.2). For double stranded nucleic acids, electrophoresis is carried out under non-denaturing conditions using a polyacrylamide or agarose matrix, depending on the size of the nucleic acid. For single stranded nucleic adds, electrophoresis should be conducted in the presence of urea, to disrupt any partially based-paired structures that may influence mobility. In both cases, the samples of interest should be run together with molar mass standards, which can either be prepared in the laboratory or purchased. 

Gel Electrophoresis of Nucleic Acids Electrophoresis in Denaturing Media... 

Abrams, E.S. and Stanton, V.P. Jr (1992) Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. Methods inEnzymology 212, 71-104. 

Myers, R.M., Fischer, S.G., Lerman, L.S. and Maniatis, T. (1985a) Nearly all base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3131-3145. 

Myers RM, Fischer SG, Maniatis T, Lerman LS. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res 1985 13(9) 3111-3129. 

Fig. 6.14. Agarose gel electrophoresis of Echinococcus granulosus (horse strain) RNA. The samples in lanes 1,2,7 and 16 were prepared in 10 mM phosphate buffer, pH 6.8, and were not denatured. The samples in lanes 3, 4, 8 and 13-15 were denatured in 50% (w/v) dimethylsulphoxide in 10 mM phosphate buffer, pH 6.8. The samples in lanes 5,6,9 and 10 12 were denatured in 1 m glyoxal/3 m urea in 10mM phosphate buffer, pH 6.8. Lanes 1,3 and 5, E. granulosus total nucleic acids (10 fig) lanes 2,4 and 6, E. granulosus RNA (5 fig) lanes 7,8 and 9, Escherichia coli RNA (10 fig) lanes 10-16, Schistosoma mansoni RNA (10 fig). The E. coli RNA was included as a molecular weight marker the larger subunit is approximately 1 000 000 and the smaller subunit is 500 000. The RNA was visualised by staining with ethidium bromide and ultraviolet illumination. (After McManus et al., 1985.)...

Khrapko K, Hanekamp JS, Thilly WG, Belenkii A, Foret F, Karger BL (1994) Constant denaturant capillary electrophoresis (CDCE) A high resolution approach to mutational analysis. Nucleic Acids Res 22 364-369. 

This heat must be dissipated by cooling, which can be done but only to a limited extent The ability to dissipate heat efficiently is usually the factor that limits the speed of electrophoresis, since excess heat leads to non-uniform electrophoresis and a decrease in resolution. The main reason for this is convection in matrix-free electrophoresis in solution, and the effect of temperature on viscosity and diffusion. High temperatures can also lead to denaturation of proteins and nucleic acids. The thinner the layer used for electrophoresis, the more readily is the heat dissipated, and the higher the voltages that can be used. The thickness of the layer will be a compromise between a desire to have a thin layer to minimise heat problems whilst maintaining sufficient capacity to ran samples that can be detected easily. Consis-... 

Northern blotting was not named for its inventor, but as a companion technique that uses RNA rather than DNA as the test nucleic acid. RNA is transferred from the gel after electrophoresis onto a solid support followed by hybridization with a specific labeled probe. Because RNA molecules have defined lengths and are much shorter than genomic DNA, it is not necessary to cleave RNA before electrophoresis. However, because of the secondary structure of RNA, it is necessary to perform electrophoresis under denaturing... 

As an alternative to transfer methods, gels in which nucleic acid is fractionated can, after drying, be submitted directly to denaturation and hybridization within the gel. Finally, fractionation by electrophoresis after a solution hybridization step (Section 12.4) or after PCR and in-gel detection is also convenient. A tracer amount of labeled precursor can be added to the PCR mix (Verbeek and Tijssen, 1991) or a 5 -labeled internal primer can be added at the end, followed by 1 PCR cycle (Parker and Burmer, 1991) and the products analyzed in the gel. Drying of the gel enhances the signal. 

DNA is denatured in the gel (RNA is denatured before electrophoresis) and the face of the gel slab is placed in contact with a membrane made of nitrocellulose, nylon, or another material. The single-stranded nucleic acid molecules are then transferred from the gel to the membrane by capillary transfer (blotting) or other methods. The membrane containing the blotted nucleic acids is called the blot. 

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