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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1/2.3 Appendix 6


TITLE: SOUTHERN BLOT PROCEDURE


PROCEDURE

  1. Using Whatman 3MM chromatography paper, prepare the 2 layers of wick.
  2. Soak the wick layers individually in 20xSSC and place over the glass bridge.
  3. Take a 24 x 24cm 3MM sheet soaked in 20 X SSC and place centrally on the bridge.

  4. Lower the plastic screen over the wick and tape into position.
  5. Dampen exposed surface with a few ml of 20xSSC (rolled across the surface with a 10ml pipette.
  6. Transfer the gel on its tray from the Neutral wash and carefully draw away the gel tray leaving the gel on the blotting surface with the wells along the top edge. Exclude air bubbles with the pipette.
  7. Cut out a 20x20cm square of nylon membrane. Along the top edge write the date and the gel number with a pencil, then cut off the top right hand corner.
  8. Wet membrane briefly in 20 x SSC and place over the gel with the labelling face down and along the well edge. Exclude air bubbles with the 10ml pipette.
  9. Cut out 4 pieces of 3MM chr paper (21x21cm). Soak these individually in 10xSSC and place over the membrane, excluding air bubbles each time.
  10. Place two stacks of 7400 paper towels along the length of the gel, so they meet in the centre and press these in place with a 20x20cm stainless steel plate.
  11. After 15-30 min move the soaked towels and replace with fresh towels this time across the gel. Pour 20 X SSC into the reservoir to a depth of at least 1cm and leave overnight. Soaked towels may then be replaced again with fresh after a further 15-30 min.

REAGENTS:

  • 20xSSC Sodium chloride 175.3g
  • Sodium citrate 88.2g
  • Distilled water 800ml
  • Adjust to pH 7.0
  • Make up to 1l with distilled water.
  • Dilute 1:1000 and check conductivity (around 450mS)
  • Autoclave and store at room temperature for a maximum of 12 months.
  • Records: Record batch numbers of 20xSSC and Hybond N on the gel blot Logsheet.
     


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

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This work cannot be reproduced in whole or in part without the express written permission of the CABRI consortium.
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