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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1/2.3 Appendix 4

 


TITLE: LOADING AND RUNNING OF ANALYTICAL GELS AND PREPARATION OF GEL LOADING MIX


INTRODUCTION

This procedure is part of the DNA fingerprinting process and describes the preparation of samples and their subsequent electrophoresis.

PROCEDURE

  1. Fill out a Gel Blot Log Sheet with all samples and Lambda HindIII control (or other appropriate marker) lane before the first and following the last sample.
  2. Into microtubes take sufficient DNA digest to contain 5mg and add 1XTBE or distilled water to make the volume up 20ml. Add 6ml loading mix B (see below) to each digest.
  3. Make up two lambda HindIII controls (or appropriate alternative):
  • 5ml lambda HindIII (0.25mg/ml)
  • 15ml 1XTBE
  • 6ml Loading mix B (see below) or gel loading solution
  1. Place the gel in a gel tank and cover with 1XTBE + ethidium bromide. Connect gel tank to power pack and briefly run at 75V.
  2. Check powerpack current -

    This should be 0.05-0.06 A. If the current is outside this range it can be corrected by disconnecting the power pack and adjusting the buffer level.

  3. Load controls and samples in the order given on the gel log with lane numbers rising from left to right on the gel (with the wells at the end of the gel furthest away from you ie the negative terminal).
  4. Replace gel tank lid and set electrophoresis at 75V and check the current. Check that the negative terminal is at the well end of the gel.
  5. Run gel electrophoresis for approximately 17h (e.g. 17.00 to 10.00)

REAGENTS

  • 10 X TBE
  • TRIZMA 162g
  • Orthoboric acid 46.3g
  • EDTA 9.5g

Make up to 1l with distilled water. Adjust pH to 8.7 (range 8.65 -8.75). Autoclave and store at room temperature for up to 6 months.

Loading Mix B:

  • Gel loading solution A 1.0ml
  • Ethidium bromide (Sigma or equivalent) 0.25ml
  • (10mg/ml in 1XTBE)
  • Electrophoresis buffer (10 X TBE) 0.25ml

Gel Loading solution A:

  • Ficoll 400 (Sigma or equivalent) 1.29g
  • Bromophenol blue (Sigma or equivalent) 0.02g
  • 10 X TBE (See above) 0.67ml

Distilled water 10ml
 


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

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