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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1.2.3 Appendix 9


TITLE: PROCESSING OF AUTORADIOGRAPHS AND SUBSEQUENT INTERPRETATION AND STORAGE


INTRODUCTION

Autoradiographs are a means of visualising the reaction between DNA fragments and probe, thus allowing the comparison of samples and providing a permanent record which may be stored and used for future reference.

PROCEDURE

NB: Steps 1 to 7 should be carried out in the dark room with all illumination switched off.

Latex gloves should be worn at all times.

Timing is facilitated by use of a luminous dial stop clock.

  1. Remove the X-ray film from the autoradiograph cassette.
  2. Place film into a bath of Photosol CD18 X-ray developing solution (or equivalent) diluted with reverse osmosis or deionised water as per manufacturers instructions.
  3. Agitate the bath to ensure that the film is fully submersed. Leave for 5 minutes.
  4. Using plastic forceps remove the film from the developing solution. Mount the film in the developing frame.
  5. Immerse the frame into the Photosol SB80 stop-bath (or equivalent), diluted as per manufacturers instructions with reverse osmosis or deionised water. Leave for 1 minute.
  6. Transfer the frame into the Photosol CF40 fixer-bath (or equivalent), diluted with reverse osmosis or deionised water as per manufacturers instructions. Leave for 1 minute, after which time the light may be switched on.
  7. Leave the frame and film in the fixer for a further 5 minutes.
  8. Remove the film from the frame and immerse the film in tap water for at least 20 minutes.
  9. Dry the film by allowing it to hang freely in an incubator, or the hot room at 37 1C. At this stage the emulsion of the film is easily scratched and the film must be handled with care, but the autoradiograph can be studied and a decision made about exposure times for further autoradiography.
  10. Leave the film in the hot room overnight to dry thoroughly.
  11. Label the autoradiograph(s) with the FP number of the original membrane, the probe and protocol used, the date of development and the exposure time. Label each lane on the autoradiograph with the DNA number of the sample and name of the cell line, together with a copy of the gel/bot log in clear, dry plastic wallets in a master file in order of gel no. (FP no.).
  12. Comparisons are made by eye between the profiles for distribution banks and master banks of the same cell line either on the same autoradiograph or with previous radiographs. The standard samples (e.g. HeLa S3 and/or K562) should be compared with previous results and authorised by the line manager.
  13. Additional techniques are available, these are:

Autoradiograph profiles may be recorded by hand on transparent sheets, copied on duplicating film (e.g. Sigma) or digitised into the Molmatch software.


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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