Gel electrophoresis is a staple method in a lot of experiments, but to take your analysis one step further, you can look into trying out Southern blotting. Southern blotting was developed by Edwin Southern in the 1970’s at Edinburgh University, and has been used regularly in laboratories since to further analyze DNA bands separated during gel electrophoresis.
What is Southern blotting and why do we do it?
Southern blotting takes the DNA from the gel slab after electrophoresis has finished running, and transfers it to a membrane. Typically the membranes are made of nylon or nitrocellulose because these have the ability to transfer the DNA from the gel to the membrane using capillary action. Then the bands on the membrane can be visualized. The reason we perform Southern blotting is to detect specific DNA molecules from other non-specific DNA molecules. Additionally, it is used when doing restriction fragment length polymorphism (RFLP) or variable number tandem repeat (VNTR) analyses. Both RFLP and VNTR can be used for forensic analysis, paternity testing, and identifying and understanding mutations in the genome. For example, fragile X syndrome and sickle cell anemia are conditions that can be diagnosed using Southern blotting. Along with this, Southern blotting can be used for sequencing DNA for both known and unknown sequences. So even though Southern blotting is a longer experiment, it has many useful applications for many scientific disciplines!
How is Southern blotting done?
Once your gel has finished running, a few preparatory steps are needed to ensure that your bands of DNA will transfer efficiently and accurately to the membrane. First, the gel has to be treated in depurination and denaturation solutions to prepare and cut the DNA bands so they can migrate to the membrane in smaller pieces. In the depurination step, acid is used to remove the purines in the DNA to cut it up. Then denaturation is done using a basic solution which essentially disrupts the bonds in the DNA. This all together helps the bands of DNA transfer to the membrane. The membrane also has to be treated with the denaturing solution before transfer. Once everything has been prepared, it can be assembled into a transfer assembly. Transfer is usually done with capillary action or can even be done with an electric current. Once the gel and the membrane are in the transfer assembly, the bands from the gel will start migrating to the membrane! Times vary for this based on the size of the bands and other factors, but on average the transfer occurs overnight. The membrane can then be fixed so that the bands do not migrate or get disrupted, this is usually done with heat or vacuum.
How do I visualize the bands on my Southern blot?
Using a stain such as Blue-Blot DNA Stain™ will allow students to visualize their DNA bands. Other common methods of observing bands are by using radio-labeled probes that can be visualized with X-ray cassettes or in some cases, UV light. Bands can be visually compared to each other to draw conclusions about the experiment, and membranes can be transferred to lab notebooks for safe-keeping and analysis.
Southern blotting isn’t the only blotting technique used by scientists! Other blotting techniques include western and northern blotting. Western blotting is used to visualize proteins while northern blotting is used to visualize RNA. All three of these blotting techniques are commonly used in labs depending on the type of experiment.
If you are interested in hands-on experience with Southern blotting, check out these EDVO-Kits:
DNA Fingerprinting with Southern Blot