![]() However, RNA molecules are very sensitive to degradation by RNA-ases, and therefore contamination of the material by this extremely stable enzyme must be avoided. The only difference with Southern blotting is that RNA molecules are transferred to the membrane. This transmission is called northern transmission. The following photo describes transfer of RNA molecules after their separation by electrophoresis. The probe will only hybridize to DNA molecules that contain a nucleotide sequence complementary to the probe sequence. After transfer, the DNA is immobilized on the membrane by drying or irradiation with UV light and hybridized with a radioactive DNA-probe containing the studied sequence. ![]() The DNA is denatured either before or during transfer by placing the gel in an alkaline solution. Such transfer of DNA is called Southern transfer. The basic feature of this method is the transfer of DNA molecules separated by gel electrophoresis to a nitrocellulose or nylon membrane. Southern published an important new procedure that made it possible to determine the location of genes and other sequences in restriction fragments separated by gel electrophoresis. Agarose gels are better sieves for large molecules (over a few hundred nucleotides), while polyacrylamide gels are better for resolving small DNA molecules.ĭNA Analysis by Southern Blotting ![]() Agarose and polyacrylamide gels act as molecular sieves that slow the passage of large molecules more than small ones. DNA molecules have essentially the same charge per unit mass, so they are separated on agarose or polyacrylamide gels almost exclusively based on their size or conformation. Gel electrophoresis is a powerful tool for separating macromolecules of different sizes and charges.
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