1.Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube). 2.Resuspend pellet with 300μl STET buffer (900μl). After resuspending add 30μl RNase/lysozyme mixture (100μl). 3.Boil for one minute 15 seconds (one minute 45 seconds). 4.Spin in microfuge for at least 15 minutes. 5.Take supernatant and phenol extract with 150μl (500μl) STET- saturated phenol. 6.Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). Let sit on ice for 5-10 minutes. 7.Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes. 8.Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube. 9.Important: Wash with 80% ethanol (95% will cause the residual Triton to precipitate) 10.Resuspend pellet in 50-200μl. Lysozyme/ RNase mixture 10mg/ml lysozyme 1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive) 50mM Tris-HCl pH8.0 Store at -20℃ in small aliquots. Do not refreeze after thawing. STET 8% sucrose 5% Triton X-100 50mM Tris-HCl (pH8.0) 50mM EDTA pH 8.0 Filter sterilize. Store at 4℃ |
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