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
