SPOT DNA Extraction from 142mm Durapore 0.22µm Filters
Prepare a boiling water bath; this MUST be a rolling boil.
Place 50ml falcon tube with 142mm filter on dry ice.
Using the bottom of an untouched STERILE 15ml polypropylene falcon tube as a pestle, vigorously crush the 142mm filter while in the 50ml falcon until it has been pulverized and the filter material is BELOW the 10ml line.
Add 9mls of 1X STE directly to the crushed filter pieces.
Vortex for 10 seconds.
Add 1ml of 10% SDS drop‐wise while swirling.
Spin briefly in Eppendorf 5810R centrifuge at 15‐25ºC by spinning up to 4000 rpm (3220xg) and immediately stopping.
Place in rolling, boiling water bath for 2 minutes.
Spin at 4000 rpm (Eppendorf 5810R) for 10 minutes at 15ºC to separate filter and debris.
Add 3mls of 10.5M NH4OAC to an Oak Ridge tube for each filter.
Pour supernatant (only) from 50ml falcon tube into Oak Ridge tube (38ml capacity).
Use 1ml pipette to transfer any remaining supernatant to Oak Ridge tube: ~0.5‐1ml.
Remove any large filter pieces using STERILE 1ml serological pipette to reach the bottom and drag the filter pieces up the sides.
Add 28mls of 200 proof (100%) EtOH (molecular grade).
Invert to mix thoroughly.
Store at ‐20°C overnight.
Pellet DNA in Sorvall RC5‐B at 4ºC using the HB‐4 rotor by spinning at max speed of 13,000RPM (27,900xg) for at least 2 hours.
Gently pour out supernatant and dry pellet upside‐down in fume hood for at least 2 hours.
Resuspend in 500ul 1X TE (pH 8.0) for 2 hours at 37ºC.
Transfer to 2ml non‐LoBind tube.
Add 500µl phenol.
Mix 5 times by gentle inversion.
Spin 2 min at 12000 rpm (13,000xg) to separate organic and inorganic phases (Beckman, beige). 
Remove and discard bottom (phenol) layer.
LEAVE INTERFACE.
Add 300µl phenol and 300µl SEVAG (CHCl3:IsoamylOH 24:1 v/v) to aqueous phase.
Mix by gentle inversion 5 times.
Spin 2 min at 12000 rpm (13,000xg).
Remove and discard bottom layer.
LEAVE INTERFACE.
Add 500µl SEVAG (CHCl3:IsoamylOH 24:1 v/v).
Mix by gentle inversion.
Spin 2 min at 12000 rpm (13,000xg).
Remove TOP layer (YOUR DNA) and transfer to new 2ml non‐LoBind tube.
LEAVE INTERFACE BEHIND.
This top layer is your DNA!!!
Add 125µl 10.5M NH4OAC.
Mix by inversion 5‐10 times.
* Add 1375 µl ice‐cold 100% EtOH (200 proof, molecular grade).
* Mix by inversion 5‐10 times.
Precipitate overnight at ‐20ºC.
Spin at max speed (approximately 13,750RPM, 12,535xg) in Beckman Microfuge E with horizontal rotor at 4ºC (cold room) for 30 minutes to pellet most DNA in the center/bottom of tube.
To pellet the rest of the DNA, spin the same tube for at least 90 minutes at 14,000 rpm (20,800xg) at 4ºC (Eppendorf 5810R) .
Gently pour off supernatant.
Dry pellet for at least 2 hrs by leaving open and upside‐down.
Resuspend 5m and CMAX samples in 50µl of 1X TE and 150m, 500m, and 890m samples in 30ul 1X TE for 2 hours at 37ºC.
Transfer all re‐suspended extract to 1.5ml Eppendorf LoBind DNA tube.
Aliquot 10µl of extract to an additional 1.5ml Eppendorf LoBind DNA tube for archiving.
After re‐suspension, immediately quantify using PICO Green (Invitrogen).
Prepare 2ng/µl dilution for a working stock using no more than 1µl of extract.
Store dilution, extract, and archive at ‐80ºC
