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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
- 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:
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.
- Vortex, briefly microfuge (10,000 to 13,000 rpm) and incubate overnight at 35 to 38oC, or 3 to 4 hr at 35 to 38°C EcoRI.
- 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).
- Add 2ml gel loading solution A (protocol AHC/1998/3/3.1/2.3 Appendix 4).
- 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).
- Photograph the gel over UV-light illumination
- Plot markers on log scale graph and record size of bands or read off result using standard samples.
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