Hello,
I am trying to do DNA extraction from mites, for molecular analysis should i store the mites in 70% or 96% ethanol?
Either way, later on when i want to crush the mites and add CTAB... do i evaporate the ethanol before i do that? do i centrifuge to collect mites in bottom of 1.5ml tube and rise with distilled water?
How do i go from let's say 10-20 mites suspended in ethanol to having the mites crushed in CTAB.. please include details.
Thank you
1. DNA (genomic) extraction:
CTAB
- 2g of CTAB (cetiltrimetilamina)
- 4 ml EDTA at 0,5 M
- 10 ml Tris-HCl at 1M and pH= 8,0
- 1g of PVP (polivinilpirrolidona)
- 25 ml NaCl (4M)
Add distilled water for a final volume of 100 ml.
• Add 400-800 μL of the buffer to each sample and incubate at 60ºC for 2 h.
• Add 1 volume of chloroform:isoamyl alcohol (24:1) and shake vigorously for 10 min (this mixture should be kept at 4ºC.
• Centrifuge at 13200 rpm’s for 10 min. Recover the aqueous phase and add 1 volume of isopropanol (should be kept at -20ºC).
• Put at -20ºC for 20 min. Centrifuge at 13200 rpm’s for 30 min. Remove the liquid and wash with 70% ethanol. Centrifuge for 5 min. at 13200 rpm’s.
• Remove the liquid, dry the pellet and add 50-100 μL of Σ water.
• Add 1 μL of RNase and incubate at 37ºC for 1 hour.
Hi, store in 96 degree ethanol and let evaporate alcool from tissues before starting DNA extraction (e.g. on a bench at RT).
Good luck!
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