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 .
