96 - well DNA Extraction Protocol from 25mm 0.2µm filters
Prepare lysis buffer .
Prepare Lysozyme & RNase Aliquot .
Prepare ProK Aliquot .
Thaw filters on ice .
Do quick spin down .
Transfer each filter to screw top , O - ringed eppendorf tube , also on ice .
Add 250 µl lysis buffer with RNase and lysozyme to each tube .
Incubate 37°C for 30 min . , rotating end over end at angle , for optimal mixing with minimal frothing .
Add 18.75 µl of Proteinase K solution ( 10 mg / ml made up in lysis buffer ) to a final conc . of 0.65 mg / ml .
Add 29.9 µl 10 % SDS to a final conc . of 1 % .
Incubate at 55°C for 2 hours , rotating end over end at angle .
Towards end of this time , turn on heat block or hyb oven to 70°C .
Put elution liquid ( Buffer AE or water ) into 70°C to preheat .
Add 300 µl Buffer AL ( = Buffer AL / E without the ethanol added ) .
Mix thoroughly by vortexing and spin down quickly .
Incubate 70°C for 10 mins .
Add 300 µl 96 - 100 % EtOH .
Mix by vortexing vigorously and spin down quickly .
Check pH of lysate , must be < 7 to get max . binding efficiency to column .
Pipet onto 96 well spin columns , making sure not to wet the rims to avoid cross contamination .
Place the 96 well column plate onto the S - block for flow through collection .
Seal plate with Airpore tape sheet Spin 5788 xg for 10 min . at 40­°C .
Discard flow through .
Place in new collection tray .
Add additional lysate if needed and repeat spin , etc .
Add 500 µl Buffer AW1 , reseal plate Spin 5788 xg for 5 min . at 40°C .
Add 500 µl Buffer AW2 , reseal plate Spin 5788 xg for 5 min . at 40°C .
To dry columns , either reseal plate with new sheet and spin 5600 xg for 15 min . at 40°C atop a new collection tray or incubate in hyb oven at 70°C for 15 min .
Transfer column plate to top of rack of " elution microtubes RS " .
Add 200 µl pre - heated 70°C Buffer AE or water , reseal plate Incubate 1 min . at RT .
Spin 5788 xg 2 min . to elute .
Repeat with second 200 µl ( will increase total yield up to 25 % ) or if you wanted to keep the column small , you could use the first 200 µl elution , heat it back to 70°C , and pass it through the column again ( will increase total yield approx . 15 % ) .
Can freeze elutants and break here overnight , or for a while , before proceeding with Final DNA clean up step , particularly if DNA is in Buffer AE which is TE , so will keep the DNA relatively stable .
Transfer the eluted DNA to the 96 well PCR purification plate ( no more than 300 µl at a time ) .
Apply vacuum at 20 inches Hg until dry ( ~ 20 - 30 min . )
Rinse DNA with 100 µl Ambion water , apply vacuum 5 - 10 min . until dry .
Rinse a second time with water if you want to make sure all Buffer TE removed .
Add 20 µl dilute TE .
Pipette up and down 20 times and transfer to a clean 96 well plate
