1. Southern blot

2. Background
a method to detect the presence of a DNA sequence in a DNA sample.
The method is named after its inventor, the British biologist Edwin Southern.
Combines agarose gel eletrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization.

3. The key to this method is hybridization.
Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA.

4. There are two important features of hybridization:
The reactions are specific-the probes will only bind to targets with a complementary sequence.
The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

5. Gel and membrane preparation Probe preparation and hybridization
Procedure
DNA extraction
DNA digestion
Agarose electrophoresis
Gel and membrane preparation
Transfer
Prehybridization
Probe preparation and hybridization
Wash
Detection

6. DNA extraction
Quality and amount of DNA is important for southern blot.
Agarose electrophoresis check DNA integrity after extraction.
High quality intact DNA should give the appearance of a single band.
Degraded material will smear downwards.
Avoid RNA, protein, endogenous nuclease contamination.
Avoid DNA degradation.

7. Enzyme digestion
Restriction endonuclease digestion of genomic DNA is different from plasmid. 1ug genomic DNA: 5-30U enzyme Determine appropriate enzyme amount and digestion time. Avoid incomplete digestion: use large volume or use high concentrated enzyme.

8. Transfer
Depurination is not required for DNA fragments <10kb in size. Do not over depurinate, 10mins(or until the bromophenol blue turns yellow) is usually sufficient for most samples.
DNA is then denatured with an alkaline solution such as NAOH,which causes the double stranded to become single-stranded.
DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.
Transferred by either electrophoresis or capillary blotting.
Avoid trapping any air bubbles during transfer.
Large DNA fragments need overnight to 24h transfer.

9. Pre-hybridization 4-5 hours
Buffer binds to areas on the blot not occupied by patient DNA.
Blocks the empty sites from being bound during hybridization.

10. Hybridization and Washing
Lower hybridization temperatures achieve lower stringency ℃ is suitable for most long probes(>100base). Stringency washes will dependent on the nature ofn probe and target to be hybridized. The lower the salt concentration, the greater the stringency. The higher the washing temperature, the greater the stringency.

11. Watch points
Using too little DNA-compromise the sensitivity of the test
Using too much DNA- poor restriction enzyme digestion
Using too high voltage setting for electrophoresis- gel to melt or appearance of artifacts
Improper blocking-high background and uninterpretable results.
Insufficient washing-high background and uninterpretable results.
Excess washing- dissociate the specific hybrids.