Extracting nucleic acids from viruses on a filter
Add 1 mL T + C lysis buffer containing 100 µg / ml proteinase K to a low - volume ( 1–3 cc ) syringe that has been fitted with a female–female Luer - Lok adapter ( the injection syringe ) .
Ensure that there is minimal air in the injection syringe - adapter assembly , then connect it to the outlet of the filter .
Connect a second low - volume syringe to the filter inlet ( the aspiration syringe ) .
Hold the filter - syringe assembly vertically with the injector syringe pushing upward from below .
Hold the filter securely to the injection syringe and gently , but firmly , push extraction buffer into the filter housing until liquid just begins to appear in the aspiration syringe .
Incubate the assembly ( filter with two syringes attached ) for 15 min at 65°C in a hybridization oven .
Allow the syringe - filter assembly to cool briefly .
Remove the extract by holding the syringe assembly vertically with the aspiration syringe underneath ( and the filter upside down ) and gently pulling on the plunger to pull the extract into the aspiration syringe .
Detach the aspiration syringe .
Transfer the extract to a microcentrifuge tube .
Chill on ice for 2–3 minutes .
Add one - half volume of MPC protein precipitation reagent ( supplied in the kit ) and vortex for 10 s .
Pellet the debris by centrifugation at 10,000g for 10 min .
Transfer the supernatant ( containing the nucleic acids ) to a sterile microcentrifuge tube ; be very careful to avoid the pellet ( containing the SDS - protein complex ) .
Transfer up to 800 µl of the sample to a fresh tube ; add 1 µl polyacryl carrier , and vortex briefly .
Add 1 µl polyacryl carrier , and vortex briefly .
Add an equal volume of 100 % isopropanol .
Mix by inverting the tube several times .
Centrifuge the sample at ≥10,000g for 15–45 min .
Decant or aspirate the supernatant .
Wash the pellet twice , each time adding 70 % ethanol , centrifuging for 1 min , and decanting ( or aspirating ) the ethanol .
Air - dry the pellet , then dissolve in 10 µL of 0.02 - filtered , sterile 0.5× TE buffer heated to 50°C .
If required , DNA or RNA can be selectively removed from the total nucleic acid precipitate by enzymatic digestion with DNase or RNase .
