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This Protocol is used if DNA digests show evidence of heavy protein contamination or incomplete digestion (e.g. uv transilluminsation of digests run in an agarose gel show marked retention of fluorescing material in the well or streaks in electrophoresis lane).

1. Add 200ml of sterile 0.2M sodium acetate pH 7.5 to the digest tube.
2. Incubate at 35 to 40^oC for 1 hour.
3. Add 3ml of Proteinase K (20mg/ml).
4. Incubate at 55 to 60^oC for 1 hour.
5. Add 100ml of Phenol/Chloroform mix.
6. Vortex briefly until the mixture is a white emulsion.
7. Centrifuge for 5 min at full speed in a microfuge (eg 13,000rpm).
8. Remove top aqueous phase to a new labelled sterile microtube.
9. Add 2 volumes of absolute alcohol.
10. Incubate at -10 to -25^oC for 1 hour.
11. Centrifuge as in step 7.
12. Remove alcohol and wash pellet with 1ml of 80% (v/v) alcohol (absolute alcohol diluted in sterile water).
13. Centrifuge for 2 min as in step 7.
14. Remove alcohol and dry pellet at 35 to 40^oC for 30 to 60 min or under an infra-red lamp (minimum distance 10cm) for 5 to 10 min.
15. Add 32ml TE buffer (10mM Tris, 1mM EDTA, pH7.5 in sterile or deionised water). If there is a very low yield of DNA (e.g. very small amount of DNA precipitate) then add 10ml (for a 20ml final digest volume) TE buffer and dissolve DNA at 50 to 60^oC for 1 hour.
16. Quantify DNA using Protocol AHC/1998/3/95.

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