Capillary Blotting of RNA and DNA Gels

Edwin M Southern, University of Oxford, Oxford, UK

Quite often the gel electrophoretic migration pattern of individual components in a DNA or RNA sample is determined not by analysing the gel but rather by analysing an image of the gel created by blotting the gel on to membrane. One of the simplest procedures is capillary blotting.

Secondary article
Article Contents
. Introduction . Step 1: Equipment and Solutions . Step 2: Procedure . Hazards . Hints and Tips

Introduction
RNA and DNA can be transferred from agarose and polyacrylamide gels to sheets of nitrocellulose or other types of absorbent membrane, such as Nylon-based membranes, in such a way as to retain the original pattern of fragments. The DNA is then xed to the membrane by baking or ultraviolet (UV) light and the membrane can then be probed with labelled DNA or RNA to detect specic sequences. This pattern can then be detected by autoradiography, phosphor imaging, uorescence or chemiluminescence. The following protocol describes general procedures for the transfer of DNA and RNA to a transfer membrane before further analysis by subsequent hybridization. Nylon membranes are represented by Hybond-N (Amersham International), which is a Nylon-66 membrane, and Zeta-Probe (Bio-Rad), which is a quaternary amine derivatized Nylon membrane. Nylon membranes oer several advantages over nitrocellulose, including higher tensile strength, durability, higher DNA-binding capacity and retention, thus allowing these membranes to be reprobed several times. Another useful feature is that the low inherent uorescence of Nylon membranes allows visualization of bands directly by UV illumination (Thurston and Saer, 1989). For DNA gels, the derivatization of Zeta-Probe allows blotting to take place in 0.4 mol L 2 1 sodium hydroxide (NaOH) considerably reducing the number of steps involved, as well as improving resolution, both by reducing diusion and by preventing reassociation during transfer. This article describes the basic blotting and hybridization procedure, as used with nitrocellulose, as a reference point. All manufacturers provide
Recipe 1 Ingredient NaCl Trisodium citrate Double distilled water to nal volume 20 SSC

their own specic manuals and these must be read before use and when changing to a new type of membrane. Dierent membranes often cost substantially dierent amounts, both to purchase and to use.

Step 1: Equipment and Solutions

Apparatus for capillary blotting
A suitable transfer apparatus is shown in Figure 1. The dimensions of the tray can be chosen to suit the size of gels used, but a deep plastic tray (27 46 cm external dimensions) will accommodate the largest gels normally used, or several smaller ones. If required, only a small portion of the surface area of the transfer apparatus need be used at one time, as long as the unused surface is covered to prevent drying, for example with a polythene sheet. In this apparatus a sheet of plate glass overlaps, and is supported by, the short ends of the tray. A sheet of thick lter paper (e.g. Whatman No. 17) overhangs the long sides of the glass so that the hanging parts, which will act as wicks, dip into the tray, which is lled with 20 SSC (1 SSC is 0.15 mol L 2 1 sodium chloride (NaCl), 0.015 mol L 2 1 sodium citrate, pH 7.0) or the appropriate buer for the membrane in use. A second sheet of the same lter paper covers the horizontal surface of the rst. This can be replaced easily from time to time if it becomes damaged or dirty.

Solutions
. 20 SSC (Recipe 1)

Final concentration (mol L 1) 3 0.3

Volume/amount 175.3 g 88.2 g 1000 mL

1. Adjust to pH 7.0 with 10N NaOH. Sterilize by autoclaving. 2. Dilute 1:10 before use to a working concentration of 2 SSC.
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Capillary Blotting of RNA and DNA Gels

Glass plate Paper towels Light filter paper Glass plate 20 SSC Membrane Gel Thick filter paper with wicks

Figure 1 Transfer apparatus.

Step 2: Procedure
1. 2. 3. Pour 20 SSC on to the surface of the transfer apparatus (Figure 1) until it is just ooded. Slide the gel on to the surface without trapping air bubbles. Surround the gel with strips of plastic or glass, of about the same thickness as the gel and about 2 mm away from it. Cut a sheet of transfer membrane 1 cm wider and 1 cm longer than the gel: wet it by oating it on 2 SSC. Rinse the transfer membrane in 2 SSC, drain it for 30 s and lay it squarely on top of the gel and its surround without trapping bubbles of air beneath it. Avoid changing the position of the transfer membrane once it has made contact with the gel. Prepare a sheet of lter paper (e.g. Whatman 3MM) as for the transfer membrane and lay this on top of the transfer membrane.

4. 5.

7. Cover the unused surface of the apparatus with polythene sheet. 8. Stack paper towels on top of the lter paper, 510 cm high. Place a glass or plastic sheet on top, and weigh this down lightly; a weight of about 100500 g is sucient, depending on the gel size. A 3-mm-thick glass sheet the same size as the gel is suitable. 9. It is usually convenient to allow transfer to take place overnight, although small DNA and RNA molecules will be transferred more quickly. 10. Remove the towels and lter paper. 11. Mark the gel position and orientation on the transfer membrane with a ballpoint pen. 12. Carefully peel the transfer membrane back from the gel. 13. Rinse it for a few minutes in 2 SSC until it is free of any pieces of agarose, etc. 14. Place the transfer membrane between pieces of thick lter paper, in a sandwich held together with paper clips. 15. Bake in a vacuum oven at 808C for 2 h, or for Nylon membranes expose to UV transilluminator. 16. The lter can be stored at 2 208C indenitely.

Hazards
The hazards associated with the chemicals/apparatus used in this protocol are detailed in Table 1.

6.

Table 1 Hazards associated with this procedure Electrophoresis Great care must be exercised when using any electrophoresis equipment, especially high-voltage or constant-current supplies. If possible, always use commercially supplied apparatus that has been designed and built to international electrical safety standards: home-made equipment is always suspect in this regard. Always check that all wiring connections are properly made and any interlocks tted are secure before switching on the power supply. Always switch o the power supply before disconnecting the apparatus. Arrange the work area to reduce the risk of water or reagents splashing on to the power pack, leads, cables or chambers. Preferably use power supplies tted with electrical earth leakage detection circuitry and automatic cut-o. Exposure to UV light can create severe DNA damage and can induce apoptosis. Always wear protecting clothes and plastic masks to protect eyes and skin! Care should be taken to avoid burns when pouring hot agarose. High-temperature ovens should be sited away from any highly volatile ammable substance. Use thermally insulated gloves or tongs with insulated handles when transferring objects into and out of a hot oven. Irritating to eyes, respiratory system and skin. Causes severe burns. Wear glasses. Eye contact: rinse immediately with plenty of water for 15 min and seek medical advice. Skin contact: immediately wash skin with soap and copious amounts of water. Ingestion: if the chemical has been conned to the mouth give large quantities of water as a mouthwash. Ensure the mouthwash is not swallowed. If the chemical has been swallowed, give about 250 mL of water to dilute it in the stomach. In severe cases, obtain medical attention. Always wear UV goggles or visor. Do not look directly at the light source in transilluminators for unnecessary periods of time even if goggles are being worn. Allow for reected UV light. Do not

Exposure to UV light Hot agarose Oven

Sodium chloride NaCl Sodium hydroxide NaOH

Ultraviolet light sources

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Capillary Blotting of RNA and DNA Gels

Table 1 (Continued) expose skin to UV illumination for unnecessary periods of time. If long periods of viewing are necessary, use a UV face visor. Ensure that the eye protection provides adequate UV absorption for the intensity and frequency of UV light being used. Long-wavelength UV is less dangerous than short-wavelength UV. All vacuum systems must be properly assembled and used to avoid the risk of implosion. Vacuum evaporation systems must contain appropriate traps for volatile substances to avoid contamination of the pump. Generally, the vacuum should be released slowly so as to avoid the risk of implosion. Take care with water pumps as backow of the water stream is a common problem if the water pressure is unreliable.

Vacuum

Hints and Tips

Step 2
2.1 It is most important to keep the transfer membrane clean; handle it only using gloves or forceps. 2.2 Note that if the surface of the apparatus is too wet, liquid bridges may form between the surround and the gel; excess liquid should be mopped up prior to laying the transfer membrane down.

2.3 The towels soak up the 20 SSC, causing it to ow through the gel, transferring the fragments on to the transfer membrane. 2.4 The completeness of transfer can be checked by restaining and photographing the gel.

References
Thurston SJ and Saer JD (1989) Ultraviolet shadowing nucleic acids on nylon membranes. Analytical Biochemistry 178: 4142.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net