Southern hybridization, a method to analyze sequences of interest, requires transfer of electrophoresed nucleic acid samples from a gel on to a solid support (i.e. membrane). The transfer can be achieved with the aid of capillary pressure, electric field or vacuum force. The capillary method is the traditional method, however, it consumes a relatively large amount of time (up to 12 hrs), buffer and blotting paper. On the other hand, electro-blotting is faster, but requires special care to prevent crushing or melting of the agarose gel. Nevertheless, electro-blotting is an efficient method for polyacrylamide gel transfer where capillary or vacuum techniques are unsuccessful due to the smaller pore size of polyacrylamide gels. Vacuum blotting employs vacuum pressure to transfer nucleic acid in the presence of a high concentration of salts.
The VacuGene XL Vacuum Blotting System executes vacuum blotting with gel treatment carried out in the blotting unit itself. Both pre-transfer and transfer steps can be carried out in the same unit. The unit has an acrylic body, which consists of a base and a frame supported on four rubber feet with four clamps to lock them together. The vacuum pump is connected through the connector on the outer of base. At the rim of base is a rubber gasket, which provides the vacuum seal on operation. A collapsible stand is provided which aids in removal of excess solution. Silicone tubing connects the blotting unit with the vacuum pump through a liquid trap. The vacuum pump provides a low-pressure vacuum in the range 0-100 mbar (± 5%).
The polyethylene screen supports the membrane, mask and gel. The polyethylene screen is placed on the inner rim of the base unit with the shiny side up. Before use, it is wet with distilled water. The activated membrane, which can be nylon, PVDF or a nitrocellulose of pore size 0.45 um or less, is placed over the wet porous screen. A window that is smaller in size than the membrane and gel is cut on the mask provided. The mask is then placed such that it covers the gasket and the window overlaps the membrane. The gel is then positioned such that it overlaps the window by 3-10 mM. The VacuGene XL Vacuum Blotting System can accommodate gels up to 20 cm x 30 cm. The mask enables the development of vacuum pressure, which is concentrated on the gel. Care is to be taken that the wells of the gel lie over the mask; otherwise, there will be vacuum leakage. Also air bubbles trapped between the screen, membrane and gel should be avoided. Afterwards the top frame is placed over the unit and tightened with the clamps. The vacuum is applied at 50 mbar and depurinaturation solution is poured over the gel for required time (generally 7 min for thin and low % agarose gels). The solution is removed with the aid of the collapsible stand. This is followed by similar treatments with denaturing (7 min), and neutralizing (7 min) solutions. Finally, transfer buffer is poured on to the gel and transfer is carried out for 30 minutes. On average, the transfer protocol is complete in 1-2 hours. Nevertheless, optimization of transfer will depend on gel thickness, percentage, sample type and vacuum applied.
We often perform Southern hybridization experiments to study the chromatin organization of cell cycle related genes. The rat epithelial cell nuclei are subjected to partial Micrococcal nuclease digestion followed by DNA purification. DNA samples are electrophoresed in a 1.8% agarose gel, blotted and finally hybridized with gene specific probes.
Besides being easy to set up, the gel treatment procedures can be carried out within the apparatus. This saves a lot of solution and time. Nevertheless, we are more comfortable with gel treatment outside the unit as it provides flexibility by avoiding standardization of treatment time coupled with vacuum pressure setting. Hence we have not tried gel treatment within the apparatus. The gels are treated in similar fashion as for capillary transfer. The solutions are in surplus hence ensuring uniform treatment. Depending upon the agarose gel percentage, transfer times are set. For a 1.8% gel, we set transfer for 90 minutes.
I generally transfer 2 gels, 15 cm x 14 cm each. Sometimes I encounter problems in placing the mask in the optimum position, probably because of large sized windows. In addition, a fold appears in the mask when the top frame is clamped. Nevertheless, I am able to reset the mask (sometimes, I have to reset many times) to the proper position and the proper vacuum is generated.
We purchased VacuGene XL Vacuum Blotting System 2 years ago; since then, setting up Southern blotting is much less tedious.
Nidhi Vishnoi
Senior Research Fellow
Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Chemical Carcinogenesis