(last update : 18-10-1999)
Extraction of RNA with CTAB and Macaloid
- Reagents needed :
- Washing buffer :
76 % ethanol and 10 mM ammoniumacetate
- CTAB : (store at room temperature)
- 2 % (w/v) CTAB (hexadecyltrimethylammoniumbromide)
- 1,4 M NaCl
- 0,2 % (v/v) 2-mercaptoethanol
- 20 mM EDTA
- 100 mM Tris/HCl pH 8,75
- 100 µg/ml proteinase K : (add just before use !!!!)
- Macaloid :
- Method :
!!!!! EVERYTHING HAS TO BE DONE ON ICE !!!!!!!
- put in a 2 ml tube with screwcap 500 µl CTAB and ± 0,1 g glass beads and 6 µl proteinase K (1 mg/100 µl)
- add a nematode drop (from a culture dropped into liquid nitrogen)
- beadbeate during 30 seconds at 5000 cycles/minute (if the ice is almost completely defrosted)
- incubate 30 minutes at 60°C and shake regularly
- add 500 µl chloroform/isoamylalcohol (24/1) and shake gently
- centrifuge 10 minutes at 8.000 rpm (4°C)
- transfer the aqueous fase into an other tube and add an equal volume cold isopropanol
- put 1 hour at -20°C
- centrifuge 15 minutes at 14.000 rpm (4°C)
- remove supernatans
- dissolve pellet in 500 µl DEPC destilled water, add 170 µl macaloid and 500 µl phenol/chlorophorm/isoamylalcohol pH 8 and shake for 1 minute
- centrifuge 15 minutes at 10.000 rpm (4°C)
- transfer the aqueous fase into an other tube
- add 500 µl DEPC destilled water, add 170 µl macaloid and 500 µl phenol/chlorophorm/isoamylalcohol pH 8 and shake for 1 minute
- centrifuge 15 minutes at 10.000 rpm (4°C)
- transfer the aqueous fase into an other tube
- add 0,1 volumes sodiumacetate pH 5 and an equal volume cold isopropanol
- put 1 hour at -20°C
- centrifuge 15 minutes at 14.000 rpm (4°C)
- remove supernatans and wash the pellet with 500 µl 75% ethanol
- add 50 µl DEPC destilled water to the pellet (to store the RNA, is is best to leave it in 75% ethanol)
- use 1 to 5 µl for RT-PCR
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