Southern Blotting (First Method)

Introduction
This method to know the presence or absence of a particular fragment in genomic DNA was first developed by E. D. Southern in 1975. The advent of Southern blotting technique was a turning point in the field of molecular biology.

It involves the capillary transfer of DNA fragments from an agarose gel to various types of membranes. Restriction Fragment Length Polymorphisms can be analyzed using the technique, wherein DNA fragments are separated on agarose gels denatured in situ and transferred onto membranes for analysis.

Materials

• Denaturation solution: NaCl, 1.5 M and NaOH, 0.5 M
• Neutralization solution: NaCl, 1.5 M; Tris-Cl (pH 7.5), 0.5 M and EDTA
  (pH 8.0), 1 mM
• 20X SSC: NaCl, 1.5 M and trisodium citrate, 0.1 M
• Depurinization solution: 0.25 N HCl
• Nylon or nitrocellulose membrane

Procedure
After agarose gel electrophoresis, photograph the gel and soak it in 0.25 N HCl for 15 minutes at room temperature, with gentle shaking.

Decant the acid solution and denature the DNA by soaking the gel in several volumes of denaturation solution for 30 minutes at room temperature, with constant shaking.

Neutralize the gel by shaking in several volumes of neutralization solution for 30 minutes at room temperature, with shaking.

Wrap a piece of Whatman 3-mm paper around a glass plate. Place the wrapped support on a large plastic tray with the ends of the 3-mm paper dipping into the 20X SSC solution in tray.

Invert the gel and place it on a damp 3-mm paper on the support. Make sure that there are no air bubbles between the 3-mm paper and the gel.

Cut a piece of nylon membrane slightly bigger than the gel. Use gloves and forceps to handle the membrane.

Float the membrane on 20X SSC until it wets completely.

Place the wet nylon membrane on top of the gel. Remove all the air bubble that are trapped between the gel and tile membrane.

Wet 2 pieces of Whatman 3-mm paper, cut to exactly the same size as the gel in 10X SSC, and place them on top of the membrane. Again remove the air bubbles.

Cut a stack of coarse filter paper just smaller than the gel size. Keep on top of the Whatman filter papers.

Put a glass plate on the top and place (about 1 kg) on it to exert pressure.

Allow the transfer of DNA to proceed for about 12–24 hours.

Remove the stack of coarse filter papers and the 3-mm paper above the gel. Turn over the dehydrated gel and membrane and lay them gel side up on a dry sheet of 3-mm paper. Mark the position of the wells on the membrane with a soft pencil. Peel off the gel. The transfer can be checked by restaining the gel. If the transfer is complete, no DNA should be retained on the gel.

Soak the membrane in 6X SSC at room temperature for a few minutes.

Allow excess fluid to drain off from the membrane and set it to dry at room temperature on a sheet of 3-mm paper.

Place the dried filter between 2 sheets of 3-mm paper.

Fix the DNA on the membrane by baking for 2 hours at 80°;C under a vacuum or cross linking on a UV transilluminator for a few minutes.

Wrap the membrane with saran wrap or keep it in an envelope made up of Whatman No. 1 filter paper and store.

Notes
Nylon membranes are preferable over nitrocellulose for transfer.

In case nitrocellulose is used, the DNA has to be fixed by baking at 80°;C for 2 hours under a vacuum. Nitrocellulose, being combustible, will become brittle if baked in the presence of oxygen.

DNA can be fixed on nylon membrane by UV-cross linking using longwave UV rays or by baking at 120°;C for 2 hours.

Care has to be taken so that the buffer passes to the filter paper through the gel only. A layer of parafilm may be put on the glass plate around the gel to avoid the filter papers touching the buffer directly.

While photographing the gel, keep a fluorescent ruler alongside the gel for proper orientation later on.

Never touch the membrane with bare hands. Any grease or powder on the membrane will prevent transfer of DNA.

Nowadays, charged and modified nylon membranes are available from Qlany suppliers, and they produce better results. Follow the manufacturer’s instructions for these membranes.

Depurinization of the DNA on the gel by using HCl is to facilitate transfer of large DNA fragments. For small DNA fragments, this step may be avoided.

Observations
Restain the gel in ethidium bromide 5 mg/mL for 45 minutes and view on a UV transilluminator after proper washings. There should not be any DNA on the gel, as the entire DNA should have been transferred to the membrane.

There will be only one band in the lane. In the case of genomic DNA, a continuous smear should be visible, as digestion will result in many pieces of varying sizes.