What you need to know about Southern and Northern blotting
Nucleic acid blotting is a well established technique for locating a gene or sequence of interest from a complex mixture of DNA or RNA. Southern and Northern blotting are the two most common nucleic acid blotting techniques. Southern blotting is used to detect a sequence from within a DNA mixture and Northern blotting detects a sequence from an RNA mixture. Dot or slot blots and colony/plaque lifts are other nucleic acid blotting methods and utilize many of the same processes.
Six steps to successful results
The nucleic acid protocol is made up of six steps:
- Nucleic acid electrophoresis
- Blotting
- Probe labeling
- Hybridization
- Detection
- Image analysis
Nucleic Acid electrophoresis and blotting
The first step of Southern and Northern blotting is to separate a mixture of DNA or RNA on an agarose gel by molecular weight. The DNA or RNA is then transferred to a membrane usually made of nylon. Either conventional capillary blotting , electroblotting or vacuum blotting (for fast and even transfer) are used as the blotting technique.
Variations of this protocol are seen when it comes to dot or slot blotting: the DNA/RNA mixture is spotted directly onto the membrane without separation. For colony or plaque lifts, you can place your membrane directly onto a Petri dish.
Labeling
Use a specific probe directed against the sequence of interest to locate the presence and, in some cases, quantify the amount of target on your blot. This probe can be labeled with radioactivity, fluorescence or tagged with an enzyme that eventually generates a chemiluminescent signal when incubated with the appropriate substrate. The choice of the label relies on many factors such as sensitivity, quantification requirements, ease of use and experimental time.
Hybridization
The probe is incubated with the blot in hybridization buffer where it hybridizes to its complementary target on the membrane. Incubation temperature and salt concentration are critical to control the stringency both during hybridization and the subsequent washes to remove unbound probes from the membrane.
Detection and Image Analysis
Once the probes are hybridized on their complementary target, one proceeds to detection. Radioactive probes are detected directly with autoradiography or via phosphorimager screens and scanners. Chemiluminescent signal can get captured on both autoradiography and imaging systems, whereas fluorescent signal can only be captured on imaging systems. Then, the acquired signal may be converted to data via image analysis software for analysis or exportation to publication media.
GE Healthcare offers you a wide variety of high-quality products for every single step of the Southern or Northern blotting protocol, giving you the opportunity to gather a complete nucleic acid blotting configuration that fits your needs.
See our product selection for each Southern and Northern blotting workflow step
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