Calibration of aTransilluminator for UVFixation of DNA to NylonFiltersEdwin 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.

Introduction

After a gel has been blotted it is necessary to fix theDNAorRNAto the filter; the only exception to this iswhenDNA isblottedon toZeta-Probemembranes in alkaline conditionsso that the denatured DNA binds irreversibly to themembrane. Baking the filter at 808C has been found to fixDNA and RNA to nitrocellulose filters, but in the case ofNylon filters the DNA can be fixed to the filter byultraviolet (UV) irradiation.

Step 1: Equipment and Solutions

Equipment

. UV light source

. Clingfilm Saran Wrap (BDH, Poole, Dorset, UK)

. Hybond-N (Amersham Life Sciences, Bucks, UK)

Solutions

. 20� SSC (Recipe 1)

Step 2: Procedure

1. Prepare 500-pg dots of denatured bacteriophage lDNAonHybond-N in 6� SSC.Use a strip of six dotsfor 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 blotshould be exposed toUV light for one specific length oftime to produce a time course. Time points of 30 s,1min, 2min, 5min, 10min and 20min are recom-mended. Ultraviolet radiation is dangerous, particu-larly to the eyes. Always wear an appropriate safetymask and ensure all skin is covered.

4. Prepare a labelled l-DNA probe by standard proce-dures; for example, nick translation or randomoligonucleotide primed labelling.

5. Hybridize the filters under standard conditions (seeAmersham International manual or equivalent).

6. After hybridization and washing, the dot blots may beexposed to film for 3 h or more as appropriate.Optimum UV exposure time may be judged by eye;however, more accurate results will be obtained if theblots are dried and the radioactivity of each dot ismeasured individually by counting in a scintillationcounter.

7. A graph of exposure time versus mean counts per stripof dots should give a clear peak; optimum exposuretime may be read off the x axis.

Hazards

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

Article Contents

Secondary article

. Introduction

. Step 1: Equipment and Solutions

. Step 2: Procedure

. Hazards

Recipe 1 20� SSC

IngredientFinal concentra-tion (mol L–1)

Volume/amount

Sodium chloride 3 175.3 gSodium citrate 0.3 88.2 gDouble-distilled

water to finalvolume

– 1000mL

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

1ENCYCLOPEDIA OF LIFE SCIENCES © 2002, John Wiley & Sons, Ltd. www.els.net

Table 1 Hazards associated with this procedure

Exposure to UV light Exposure to UV light can create severe DNAdamage and caninduce apoptosis. Always wear protecting clothes andplastic masks to protect eyes and skin!

Sodium chlorideNaCl

Irritating to eyes, respiratory system and skin.

Sodium hydroxideNaOH

Causes severe burns. Wear glasses. Eye contact: rinse imme-diately with plenty of water for 15min and seek medicaladvice. Skin contact: immediately wash skin with soap andcopious amounts of water. Ingestion: if the chemical hasbeen confined to the mouth give large quantities of water asa mouthwash. Ensure the mouthwash is not swallowed. Ifthe chemical has been swallowed, give about 250mL ofwater to dilute it in the stomach. In severe cases, obtainmedical attention.

Ultraviolet light sources Always wear UV goggles or visor. Do not look directly at thelight source in transilluminators for unnecessary periods oftime even if goggles are being worn. Allow for reflected UVlight. Do not expose skin to UV illumination for unneces-sary periods of time. If long periods of viewing arenecessary, use a UV face visor. Ensure that the eyeprotection provides adequate UV absorption for theintensity and frequency of UV light being used. Long-wavelength UV is less dangerous than short-wavelengthUV.

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

2