Southern Blotting

목차

Ⅰ Introduction
Ⅱ Materials
Ⅲ Protocol
Ⅳ References

본문내용

Ⅰ Introduction
Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern transfer and hybridization is used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA.
This protocol describes the first stages of Southern blotting : digestion of genomic DNA with restriction enzymes, separation of the resulting fragments by gel electrophoresis, and capillary transfer of the denatured fragments to a membrane.
The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1pg of the DNA for which you are probing.

Ⅱ Materials
Buffers and Solutions
Alkaline transfer buffer (for alkaline transfer to nylon membranes) : 0.4 M NaOH, 1 M NaCl
Denaturation solution (for neutral transfer) : 1.5 M NaCl, 0.5 M NaOH
Depurination solution (optional) : 0.2 M HCl
Neutralization buffer I (for transfer to uncharged membranes) : 1 M Tris (pH 7.4), 1.5M NaCl
Neutralization buffer II (for alkaline transfer to nylon membranes) : 0.5 M Tris-Cl (pH 7.2),

참고 자료

Cold Spring Harbor Laboratory. 2001. Southern blotting : Capillary transfer of DNA to membranes.
In Molecular cloning 3rd ed. (eds. Sambrook J. & Russell D.W.), pp. 6.39-6.46. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Cold Spring Harbor Laboratory. 2004. Southern blotting : Capillary transfer of DNA to membranes. Nat. Methods 1: 91-92.
Southern E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.
Reed K.C. & Mann D.A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13: 7207–7221.