Southern Blotting, Gel Electrophoresed, DNA Bands, Glass Plate, Alkali Solution, Nitrocellulose Buffer Saturated Fliter Paper

Home >> Biotechnology and Genomics >> Chimeric DNA Molecular Probes and Gene Libraries >> Southern Blotting

Your Ad Here	Southern Blotting For Southern blotting, DNA sample is first digested with a restriction enzyme and digested sample is gel electrophoresed. The DNA bands in the gel are denatured into single strands with the help of an alkali solution. Subsequently, the gel is laid on top of a buffer saturated filter paper, placed on a solid support (e.g. glass plate), with its two edges immersed in the buffer. A sheet of nitrocelluslose membrane is placed on top of the gel and a stack of many papers (paper towels) on top of this i membrane.
A weight of about 0.5 kg is placed on top of paper towels. The buffer solution is drawn up by filter paper wick, and passes through the gel to the nitrocellulose membrane and finally to the paper towels. While passing through the gel, the buffer carries with it single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels. After leaving this arrangement for a few hours or overnight, paper towels are removed and discarded. The nitrocellulose membrane with single stranded DNA bands blotted on to it, is baked at 80.C for 2-3 hours to fix the DNA permanently on the membrane.
This membrane now has a replica of DNA bands from agrose gel, and can be used for hybridization with radioactively labelled DNA or RNA probe. The membrane may then be washed to remove any unbound DNA and X-ray film is exposed to the hybridized membrane to get autoradiographs. The above steps involved in Southern blotting.	Your Ad Here

Southern Blotting Method of Analyzing DNA Segments that share Homology with a Nucleic Acid Probe