Division (a) Method according to Pastrik (2000)F1
United Kingdom
- 1))
Pipette 220 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) into a 1,5 ml Eppendorf tube.
- 2))
Add 100 µl sample extract and place in a heating block or water bath at 95 °C for 10 min.
- 3))
Put tube on ice for 5 min.
- 4))
Add 80 µl Lysozyme stock solution (50 mg Lysozyme per ml in 10 mM Tris HCl, pH 8,0) and incubate at 37 °C for 30 min.
- 5))
Add 220 µl of Easy DNA ® solution A (Invitrogen), mix well by vortexing and incubate at 65 °C for 30 min.
- 6))
Add 100 µl of Easy DNA ® solution B (Invitrogen), vortex vigorously until the precipitate runs freely in the tube and the sample is uniformly viscous.
- 7))
Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is homogeneous.
- 8))
Centrifuge at 15 000 g for 20 min at 4 °C to separate phases and form the interphase.
- 9))
Transfer the upper phase into a fresh Eppendorf tube.
- 10))
Add 1 ml of 100 % ethanol ( -20 °C) vortex briefly and incubate on ice for 10 min.
- 11))
Centrifuge at 15 000 g for 20 min at 4 °C and remove ethanol from pellet.
- 12))
Add 500 µl 80 % ethanol ( -20 °C) and mix by inverting the tube.
- 13))
Centrifuge at 15 000 g for 10 min at 4 °C, save the pellet and remove ethanol.
- 14))
Allow the pellet to dry in air or in a DNA speed vac.
- 15))
Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20 minutes.
- 16))
Store at -20 °C until required for PCR.
- 17))
Spin down any white precipitate by centrifugation and use 5 µl of the supernatant containing DNA for the PCR.