DNA/RNA Extractions from Sterivex Filters
Thaw sterivex filters on ice.
Add 40µL lysozyme solution (add 2 mg lysozyme to 40 µl Lysis Buffer) to filter.
Incubate at 37°C while rotating for 45 minutes.
Add 100µL Proteinase K solution (1mg Proteinase K in 100 µl Lysis Buffer) and 100µL 20% SDS.
Incubate at 55°C while rotating for 2 hours.
Transfer lysate to a 15 mL conical tube using a sterile 3 mL syringe.
Add 1 mL Lysis Buffer to filter and wash at 55°C for 15 minutes.
Pool with above lysate.
Add 3 mL Phenol:Chloroform:IAA (25:24:1; pH 8.0).
Vortex for 10 seconds.
Spin for 5 minutes at 2500g (speed 8).
Carefully transfer aqueous phase to a new 15 mL conical tube.
Add 3 mL Chloroform:IAA (24:1) Vortex for 10 seconds.
Spin for 5 minutes at 2500g (speed 8).
Carefully transfer aqueous phase to a centricon 100.
All of the remaining volume should be transferred to an epi tube.
Spin Centricon at 1000xg (speed4) for 20 minutes.
Add remaining volume from epi tube to centricon.
Spin until only 100µL - 500µL of aqueous phase is left in Centricon.
Add 1 mL TE Buffer Repeat step 20.
Spin until only 100 µL - 150 µL left in Centricon.
Carefully remove liquid without damaging the membrane.
Wash membrane well with 40µL of TE Buffer Pool the membrane in the same epi tube.
Note the total volume in the epi tube.
