Calibration of a Transilluminator for UV Fixation of DNA to Nylon Filters

Edwin M Southern, University of Oxford, Oxford, UK

One method of fixing DNA to filters prior to hybridization analysis is to use ultraviolet light to crosslink the DNA to the filter.

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

Introduction
After a gel has been blotted it is necessary to x the DNA or RNA to the lter; the only exception to this is when DNA is blotted on to Zeta-Probe membranes in alkaline conditions so that the denatured DNA binds irreversibly to the membrane. Baking the lter at 808C has been found to x DNA and RNA to nitrocellulose lters, but in the case of Nylon lters the DNA can be xed to the lter by ultraviolet (UV) irradiation.

Step 2: Procedure
1. Prepare 500-pg dots of denatured bacteriophage l DNA on Hybond-N in 6 SSC. Use a strip of six dots for each chosen UV exposure time. 2. Allow the DNA dots to dry at room temperature. 3. Wrap the blots separately in Saran Wrap and place, DNA side down, on the transilluminator. Each blot should be exposed to UV light for one specic length of time to produce a time course. Time points of 30 s, 1 min, 2 min, 5 min, 10 min and 20 min are recommended. Ultraviolet radiation is dangerous, particularly to the eyes. Always wear an appropriate safety mask and ensure all skin is covered. 4. Prepare a labelled l-DNA probe by standard procedures; for example, nick translation or random oligonucleotide primed labelling. 5. Hybridize the lters under standard conditions (see Amersham International manual or equivalent). 6. After hybridization and washing, the dot blots may be exposed to lm for 3 h or more as appropriate. Optimum UV exposure time may be judged by eye; however, more accurate results will be obtained if the blots are dried and the radioactivity of each dot is measured individually by counting in a scintillation counter. 7. A graph of exposure time versus mean counts per strip of dots should give a clear peak; optimum exposure time may be read o the x axis.

Step 1: Equipment and Solutions

Equipment
. UV light source . Clinglm Saran Wrap (BDH, Poole, Dorset, UK) . Hybond-N (Amersham Life Sciences, Bucks, UK)

Solutions
. 20 SSC (Recipe 1)

Recipe 1 Ingredient

20 SSC Final concentration (mol L 1) 3 0.3 Volume/ amount 175.3 g 88.2 g 1000 mL

Sodium chloride Sodium citrate Double-distilled water to nal volume

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

1. Make volume up to 900 mL. 2. Adjust pH to 7.0 with a few drops of 10N sodium hydroxide (NaOH) before adjusting volume to 1000 mL.

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

Calibration of a Transilluminator for UV Fixation of DNA to Nylon Filters

Table 1 Hazards associated with this procedure Exposure to UV light 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! 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 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. Longwavelength UV is less dangerous than short-wavelength UV.

Sodium chloride NaCl Sodium hydroxide NaOH

Ultraviolet light sources

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