Heat a thermal block to 56ºC.
Shake AL buffer before using it and if there is any precipitate, dissolve it in a bath at 56ºC.
Prepare 350 μl of negative extraction control from a pool of negative plasmas for this virus.
Pipette 350 μl of HBV plasma sample and positive and negative controls into 1.5ml microtubes.
Add 35 μl of the reconstituted protease into the microtubes.
Homogenize the AL buffer and add 350 μl to the same microtubes.
Vortex intermittently for 15 seconds; to promote efficient lysis it is essential that the sample and the AL buffer are mixed until a homogeneous solution is obtained.
Add 3.5 μl carrier DNA to the cap of each tube, close the tubes and mix by inversion.
Note: carrier DNA enables viral DNA binding to the column membrane on the next step. It is not provided in the kit. Here it was used Polyadenylic Acid, PolyA, 10 mg/ml; Amersham; It should be stored at -20°C.
Vortex intermittently for 15 seconds and spin the microtubes in the centrifuge for approximately 4 seconds.
Incubate the tubes in the thermal block at 56ºC for 10 minutes; the amount of DNA released is maximum after lysis during this incubation step. Longer incubation periods have no effect on the extraction or quality of purified DNA.
Spin the microtubes in the centrifuge for approximately 4 seconds
Add 402.5μl 96-100% ethanol, vortex intermittently for 15 seconds, and then spin in the centrifuge.
Apply the mixture from the previous step (630μl at a time), carefully, in a QIAamp Mini column incorporated into a 2ml collection tube, without letting the mixture reach the edge of the column, close the lid, and centrifuge at 6000g for 1 minute.
Place the column in a 2ml new collection tube and discard the tube containing the filtrate.
Repeat the two previous steps.
Add 500μl of buffer AW1, close the lid, and centrifuge at 6000g for 1 minute.
Place the column in a new 2ml collection tube and discard the tube containing the filtrate.
Add 500μl of buffer AW2, close the column cover, and centrifuge at 20000g for 3 minutes.
Place the column in a 2ml microtube (not supplied in the kit) and discard the tube containing the filtrate.
Centrifuge at 20000g for 1 minute to completely dry the membrane.
Discard the 2 ml tube containing the filtrate and introduce the column into a previously identified 2 ml screw tube for elution of virus DNA.
Apply 60μl of buffer AE in the center of the membrane, close the column lid, and incubate at room temperature for 5 minutes.
Centrifuge at 6000g for 1 minute.
Samples can be stored for 24 hours at 2-8°C, at -20°C for longer periods, or can be used immediately.