Introduction
Agarose gel electrophoresis for DNA
Agarose gel electrophoresis for RNA
Polyacrylamide gel electrophoresis for DNA
Reference
Electrophoresis is a method of separation and purification of macromolecules such as nucleic acids (DNA and RNA) and proteins based on the net charge, size, and conformation on a matrix. Nucleic acids have an overall negative charge due to the presence of phosphate backbone. Therefore, they move towards the anode at the migration rate that depends solely on their size. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards a pH called isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform charge, it is possible to separate them based on the size. The macromolecules are electrophoresed within a matrix or gel made up of agarose or polyacrylamide. The gels made of these polymers contain pores through which the proteins and nucleic acids can pass when voltage is applied.
Agarose is a polysaccharide extracted from seaweed and is used typically at concentrations 0.5 – 2% for electrophoresis of DNA and RNA. It forms a lattice with suitable pore size that allows the movement of nucleic acids to the positive electrode.
Figure 1. Agarose gel electrophoresis of nucleic acids
Sigma-Aldrich offers precast agarose gels in 8-, 20- and 24-well format with added ethidium bromide. Continue to “Running the gel” step if precast gels are being used.
Alternatively, cast your own agarose gels using the following procedure.
Sigma-Aldrich offers GenElute™ kits for isolation of DNA from plants and fungi (E5038), mammalian cells or tissue (G1N70, G1N10 and G1N350) and blood (NA2010 and NA2020).
To protect the isolated DNA from degradation, it is recommended that the DNA be solubilized in TE buffer (T9285).
Additionally, DNAstable® kits (93000-001-1EA, 93021-001-1EA, 53091-016-2ML and 93121-017-1EA) may be used in case the DNA is being shipped or for storage and stabilization of DNA at room temperature.
Gels incorporated with ethidium bromide:
Gels not incorporated with ethidium bromide:
Sigma-Aldrich offers Nancy-520 (01494), a fluorescent stain that can be used in the place of ethidium bromide. It is a safer, stable and more environmental-friendly alternative to ethidium bromide. Nancy-520 has an excitation wavelength of 520 nm and an emission wavelength of 560 nm.
Gels incorporated with Nancy-520:
Gels not incorporated with Nancy-520:
Figure 2.Horizontal Electrophoresis System
The quality of RNA can be assessed by agarose gel electrophoresis that resolves RNA based on the size and integrity. However, RNA forms various secondary structures due to extensive intramolecular base pairing that interferes with size-based migration on the agarose gel. Therefore, the RNA molecules must be denatured using formamide and formaldehyde.
Ensure that all the equipment used is RNAses free and all the buffers used are made in RNase-free water.
Sigma-Aldrich offers RNA sample buffers with (R1386) or without ethidium bromide (R4268)
Sigma-Aldrich offers precast 1.25% agarose gel for RNA (P6222) in 8-well format. This precast gel does not contain ethidium bromide. Continue to “Running the gel” step if precast gels are being used.
Alternatively, cast your own agarose gels using the following procedure.
Gels incorporated with ethidium bromide:
Gels not incorporated with ethidium bromide:
Polyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix. Acrylamide gels can separate DNA fragments that differ by even 0.2% in length. While proteins must be denatured by SDS before separation on polyacrylamide, DNA molecules being negatively charged need not be denatured. In comparison with agarose gels, polyacrylamide gels can accommodate larger amounts of samples and provide high resolution of DNA fragments.
Although precast PAGE gels for DNA are popularly used the cost involved is high if PAGE is a regular procedure in the laboratory. The skilled technician can prepare PAGE gels with little effort at a fraction of cost of precast gels. PAGE gels prepared in-house provide better resolution and aid in achieving consistent results.
Vertical electrophoresis chamber with power supply, glass plates, spacers and combs
*TEMED must be the last ingredient added.
If 35S or 33P nucleotides are incorporated in the oligonucleotides, follow the procedure below:
Figure 3.Vertical Electrophoresis System
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