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Description :
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
Summary of procedure
1. Extract and purify DNA from cells
2. DNA is restricted with enzymes
3. Separated by electrophoresis
4. Denature DNA
5. Transfer to nitrocellulose paper
6. Add labeled probe for hybridization to take place
7. Wash off unbound probe
8. Autoradiograph
Step 1: DNA purification
Isolate the DNA in question from the rest of the cellular material in the nucleus.
Incubate specimen with detergent to promote cell lysis.
Cell Lysis frees cellular proteins and DNA.
Proteins are enzymatically degraded by incubation with proteinase.DNA is purified from solution by alcohol precipitation.
Step 2 : Restriction digestion
Cut the DNA into different sized fragments using restriction endonucleases (RE).
Step 3 : Gel Electrophoresis
Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.
Step 4 & 5 : Denaturation & blotting
DNA is then denatured with an alkaline solution such as NAOH. This causes the double stranded to become single-stranded.
The process of transferring the DNA from the gel to a membrane is called as blotting. The blot is usually done on a sheet of nitrocellulose paper or nylon. DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe. Transferred by either electroblotting or capillary blotting.
The blot is made permanent either by:
Drying at ~80°C
Exposing to UV irradiation
Step 6 : Hybridization
The labelled probe is added to the membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.
Preparing the probe
Small piece of labelled DNA used to find another piece of DNA usually prepared by making a radioactive copy of a DNA fragment.
21 сен 2024
