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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.1/3.1


TITLE: CONFIRMATION OF SPECIES BY DIRECT ANALYSIS OF RESTRICTION DIGESTS OF GENOMIC DNA


PROCEDURE

  1. Quantify DNA samples (protocol AHC/1998/3/3.1/2.3 Appendix 11) and prepare digests of 2mg DNA (x=volume containing 2mg DNA) with HinfI, HaeIII or EcoRI as follows:
  2. xml (2mg) DNA + 2ml Enzyme (10u/m1) + 2ml 10x Enzyme buffer + (16-x)ml Sterile Water

    = 20ml final digest volume

    NB: Prepare digests of DNA's of known species in parallel.

  3. Vortex, briefly microfuge (10,000 to 13,000 rpm) and incubate overnight at 35 to 38oC, or 3 to 4 hr at 35 to 38C EcoRI.
  4. Prepare a minigel (1.0% agarose in 1 x TBE containing 10ml 10mg/ml of ethidium bromide per 100ml). (protocol AHC/1998/3/3.1/2.3 Appendix 3).
  5. Add 2ml gel loading solution A (protocol AHC/1998/3/3.1/2.3 Appendix 4).
  6. Electrophorese digests, for 30 min to 2 hr at 75V in parallel with standard digests and markers (eg l Hind III digest or BCL VI marker).
  7. Photograph the gel over UV-light illumination
  8. Plot markers on log scale graph and record size of bands or read off result using standard samples.

 


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

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