Southern Blotting Steps:
(Southern Blotting Steps from Resources 2-11)

First, DNA which can be either a single gene or of the total human genome is digested with a restriction enzyme, often restriction endonuclease. Then it is followed by separation, using gel electrophoresis (usually an agarose gel is used). The purpose of gel electrophoresis is to separate DNA fragments – the smallest DNA fragments migrate the most distance. The next step involves denaturing the DNA with sodium hydroxide (NaOH) or another alkaline/basic chemical substance. The DNA is now in single strands, rather than in the shape of the double helix.

Subsequently, the single-stranded DNA is transferred to a nitrocellulose membrane, also referred to as nitrocellulose paper or filter, which is placed on the gel. This can also be referred to as a nylon membrane. This membrane is unique to the southern blot procedure. Weight is put on top of the membrane; this is usually a stack of paper towels. The purpose of the paper towels over the filter is for DNA to participate in a process known as capillary action, paper towels absorb the alkaline chemical substance, and this binds to the single-stranded DNA. This process, unfortunately, may take several hours.

After this step, the membrane is put under ultraviolet light until baked – this is an important step to permanently fixate the DNA into the nitrocellulose membrane.

The next step is placing the filter fixated with the DNA fragments in a sealed plastic bag (see below picture) which has a radioactive hybridization probe, made of single-stranded DNA or sometimes RNA. This probe is used to form the base pairs needed to pair up with the single-stranded DNA to reform the DNA double helix. The hybridization probe is tagged using radioactivity, fluorescence, or a chromogenic dye. Enzymes can also be attached to the probe (examples include horseradish peroxidase or alkaline phosphatase).

Following this step, any excess probe is cleaned off the membrane. Then, hybridization patterns are viewed on x-ray film by a process known as autoradiography, if a radioactive or fluorescent probe is used. However, if using chromogenics, the membrane is color-developed for detection.

The best part of this procedure comes next – examining the results – the probe is used to determine which DNA fragments separated by gel electrophoresis has a specific sequence of DNA.


Image is posted with permission from Alberts et al. (2004).

Image is posted with permission from Alberts et al. (2004).

Here is a link to a great animation on the Southern Blotting procedure from the Mcgraw-Hill Company!
Mcgraw-Hill: Biology 7th Edition, Ch.16 Animation


  1. Alberts, B. et. Al. (2004). Southern blotting: gel transfer. Essential Cell Biology, 2/e, Fig. 10-14. Retrieved from Access Excellence. http://www.accessexcellence.org/RC/VL/GG/ecb/southern_blotting.php.
  2. Attias, A., & Kleinberger-Doron, N. (2006). Southern blot. Following genes and their expression. Retrieved from lecture notes online website: http://teachline.ls.huji.ac.il/72320/methods-tutorial/southern/southern-blot.html
  3. Blotting Analysis. (2009). Science Encyclopedia, Vol 1. Net Industries. http://science.jrank.org/pages/971/Blotting-Analysis.html
  4. Brinton, K., & Lieberman, K. (1994). Southern Blot. Basics of DNA fingerprinting. http://protist.biology.washington.edu/fingerprint/blot.html.
  5. Campbell, M. (2001). Southern blot method. Retrieved from lecture notes online: http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html
  6. Capillary Action. (2009). Science Encyclopedia, Vol 1. Net Industries. http://science.jrank.org/pages/1182/Capillary-Action.html
  7. Carr, S.M. (2006). Analysis of DNA by the southern blot technique. Retrieved from lecture notes online: http://www.mun.ca/biology/scarr/Gr12-18.html.
  8. Carr, S.M. (2008). Southern Blot analysis of DNA. Retrieved from lecture notes online: http://www.mun.ca/biology/scarr/Southern_Blot_analysis.html.
  9. Chemistry Daily. (2007). Southern blot. Chemistry Daily: The Chemistry Encyclopedia. http://www.chemistrydaily.com/chemistry/Southern_blot.
  10. Khalsa, G. (n.d.). Southern blotting. Mama Ji’s Molecular Kitchen. http://askabiologist.asu.edu/expstuff/mamajis/index.html
  11. Lutz, E. (2003). Recombinant DNA technology: southern/northern analysis. Retrieved from lecture notes online: http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#southerns.

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