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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1.2.3 Appendix 4


TITLE: PRETREATMENT OF FINGERPRINT GELS FOR SOUTHERN BLOTTING


PROCEDURE

  1. Following 15-19 hours electrophoresis, record the voltage, current, and time on the gel blot log sheet.
  2. Disconnect the power, pack and transfer the gel onto the transilluminator. Measure the distance travelled by the 2.3kb marker and calculate the additional electrophoresis time required for the 2.3kb marker to reach 20cm. Return the gel to the electrophoresis tank and electrophorese for the calculated period at 75v as before. When the 2.3kb marker has reached 20cm disconnect and transfer gel to transilluminator. Photograph gel. Record all migration distances of all the markers. Also record all calculations (including migration rate in cm/hr of electrophoresis) on the gel blot log.
  3. Carefully slide the gel off the gel tray onto a flat perspex sheet in a 'developing' tray.
  4. Pour 1l of Acid gel wash into the white 'developing' tray on the agitator platform.
  5. Agitate for 15 min at 100rpm.
  6. Without distorting the gel, draw off the Acid wash preferably using a force pump and 10ml pipette.
  7. Add 1l of Alkali wash to the 'developing' tray and agitate for 30 min at 100rpm.
  8. Remove the Alkali wash (as in 6).
  9. Add 1l of Neutral wash and agitate for 30 min at 100 rpm (during this time the materials for the Southern blot can be set up AHC/1998/3/3.1/2.3 Appendix 6).

REAGENTS

  • Acid wash 0.25M Hydrochloric acid 25ml of concentrated hydrochloric acid
  • acid made up to 1l with distilled water
  • Alkali wash 1.5M Sodium chloride 87.7g/1
  • 0.5M Sodium hydroxide 20.0g/1
  • Distilled water up to 1l
  • Neutral wash 3M Sodium Chloride 175.3g/1
  • 0.5M Trizma base 60.6g/1
  • Distilled water up to 800 ml
  • Adjust to pH 7.5
  • Make up to 1l with distilled water

     


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

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Site maintained by Paolo Romano. Last revised on April 2013.