(last update : 18-10-1999)

Extraction of RNA with CTAB and Macaloid


  1. Reagents needed :
  2. Method :
  3. !!!!! EVERYTHING HAS TO BE DONE ON ICE !!!!!!!

    1. put in a 2 ml tube with screwcap 500 l CTAB and ± 0,1 g glass beads and 6 l proteinase K (1 mg/100 l)
    2. add a nematode drop (from a culture dropped into liquid nitrogen)
    3. beadbeate during 30 seconds at 5000 cycles/minute (if the ice is almost completely defrosted)
    4. incubate 30 minutes at 60C and shake regularly
    5. add 500 l chloroform/isoamylalcohol (24/1) and shake gently
    6. centrifuge 10 minutes at 8.000 rpm (4C)
    7. transfer the aqueous fase into an other tube and add an equal volume cold isopropanol
    8. put 1 hour at -20C
    9. centrifuge 15 minutes at 14.000 rpm (4C)
    10. remove supernatans
    11. dissolve pellet in 500 l DEPC destilled water, add 170 l macaloid and 500 l phenol/chlorophorm/isoamylalcohol pH 8 and shake for 1 minute
    12. centrifuge 15 minutes at 10.000 rpm (4C)
    13. transfer the aqueous fase into an other tube
    14. add 500 l DEPC destilled water, add 170 l macaloid and 500 l phenol/chlorophorm/isoamylalcohol pH 8 and shake for 1 minute
    15. centrifuge 15 minutes at 10.000 rpm (4C)
    16. transfer the aqueous fase into an other tube
    17. add 0,1 volumes sodiumacetate pH 5 and an equal volume cold isopropanol
    18. put 1 hour at -20C
    19. centrifuge 15 minutes at 14.000 rpm (4C)
    20. remove supernatans and wash the pellet with 500 l 75% ethanol
    21. add 50 l DEPC destilled water to the pellet (to store the RNA, is is best to leave it in 75% ethanol)
    22. use 1 to 5 l for RT-PCR


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