Southern blot analysis reveals information about DNA identity, size, and abundance. It is a classic technique that involves separating DNA fragments based on size via electrophoresis, transferring them to a membrane, hybridization with a labeled sequence-specific probe, washing, and finally detection of labeled DNA band(s). Life Technologies offers one of the most comprehensive portfolios of products for Southern blot analysis.

From reliable restriction enzymes to the fast, convenient E-Gel® agarose gel electrophoresis system and BrightStar®-Plus membranes, our products not only help meet your Southern blotting analysis needs, they also speed up the process.

Step-by-Step Guide to Southern Blot Analysis

Step 1: DNA digestion

Obtaining complete fragmentation of your DNA at the intended restriction enzyme sites is a critical step in Southern blot analysis. Life Technologies offers a wide selection of high-quality restriction enzymes so that you can restriction digest your DNA to help provide clear, unambiguous Southern blot data. Each restriction enzyme has been validated for use with one of our universal buffers (L, M, H, K, or T(+BSA)) and is supplied with the recommended buffer.

 

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Step 2: Gel electrophoresis

Fragmented DNA is typically electrophoresed on an agarose gel to separate the fragments according to their molecular weights. Acrylamide gels can alternatively be used for good resolution of smaller DNA fragments (<800 bp).

The E-Gel® agarose gel electrophoresis system is ideal for rapid, high-resolution electrophoresis of restriction digests, PCR reactions, and plasmid preparations. The system includes gel running bases, plus data visualization and capture equipment. It uses E-Gel® precast gels that are supplied ready to use with electrodes and nucleic acid stain. There are no gels to pour, no buffer to make, and no gel boxes to assemble. Just load your DNA samples and run. Learn more about the E-Gel® agarose gel electrophoresis system.

For determination of DNA size, a wide range of DNA ladders are available for accurate size and mass estimations, including 100 bp ladders and 1 Kb Plus ladders. Learn more about DNA ladders  for gel electrophoresis.

 

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Step 3: Blotting

After electrophoresis, DNA is transferred to a positively charged nylon membrane. Traditional transfer of DNA is done overnight using an upward-transfer method. For reliable and consistent transfer with minimal background, BrightStar®-Plus Positively Charged Nylon Membranes are highly recommended. The membranes are ideal for use with radiolabeled and nonisotopic probes to achieve maximum hybridization signal.

For fast, reproducible transfer, the iBlot® 7-Min Blot  offers complete transfer of DNA to the nylon membrane in seven minutes. With the iBlot® system, there is no need for additional buffer or liquids, which can add variability in the results.

 

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Step 4: Probe labeling

A nucleic acid probe with sequence homologous to the target sequence under study is labeled with radioactivity, fluorescent dye, or an enzyme that can generate a chemiluminescent signal when incubated with the appropriate substrate. The choice of the label depends on several factors such as the nature of your probe or probe template, sensitivity needed, quantification requirements, ease of use, and experimental time.

 

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Step 5: Hybridization & washing

During hybridization, the labeled probe is incubated with the DNA fragments that are immobilized on the blot under conditions that promote hybridization of complementary sequences. When used for both prehybridization and hybridization, ULTRAhyb® Ultrasensitive Hybridization Buffer, can increase sensitivity up to 100 times compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 target molecules can be detected. Because ULTRAhyb® buffer maximizes blot sensitivity, for many targets hybridization can typically be performed in just 2 hours.

After hybridization, the unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High-stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized probe molecules. The result is that only fully hybridized labeled probe molecules, with complementary sequence to the region of interest, remain bound.

 

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Step 6: Detection

In the detection step, the bound, labeled probe is detected using the method required for the particular label used. For example, radiolabeled probes may be detected using X-ray film or a phosphorimaging instrument, and enzymatically labeled probes are typicallly detected by incubating with a chemiluminescent substrate and exposing the blot to X-ray film.

The BrightStar® BioDetect™ Kit includes reagents and materials for detection of biotinylated DNA probes. This complete kit has been optimized for use with BrightStar®-Plus Membranes and offers low-background, high sensitivity, nonisotopic detection compatible with Southern, Northern and dot blots. The BrightStar® BioDetectTM Kit takes advantage of one of the brightest and longest-lived chemiluminescent substrates, CDP-Star® substrate, that provide maximum sensitivity and the option of multiple exposures over a two-day time frame.

 

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For Research Use Only. Not for human or animal therapeutic or diagnostic use.