Southern Blot Steps
- DNA is isolated and cut with restriction enzymes.
- The DNA fragments are then analyzed by gel electrophoresis and separated by size (see previous blog post on Separation and Detection).
- Depurination – Gel is soaked in hydrogen chloride (HCl) to remove the purine bases from the sugar-phosphate backbone. This loosens up larger fragments before denaturation.
- Denaturation – The DNA is denatured by exposing the gel to sodium hydroxide (NaOH). Denaturation breaks the hydrogen bonds that hold the DNA strands together.
- Blotting – The denatured DNA is transferred to a solid substrate (nitrocellulose) that helps to facilitate probe binding and signal detection.
- Pre-hybridization – Prevents non-specific binding of the probe to other sites on the membrane surface.
- The membrane is exposed to the hybridization probe, usually a single DNA fragment with a specific sequence to the target DNA. The probe DNA is labelled either with radioactivity or fluorescent dyes.
Importance of the Membrane
Nitrocellulose and nylon membranes are best for smaller sized single stranded DNA fragments. It is compatible with many types of buffers and transfer systems. These membranes work well with protein and nucleic acids.
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