DNA Extraction from sorted cells
Prepare 2x lysis buffer and add to the volume of sample so that its final concentration is 1x .
Filter - sterilize through 0.2 μm .
Prepare 2x Lysis buffer with Lysozyme & RNase ( 1 ) : right before use add to 1 ml aliquot of lysis buffer :
Shake to dissolve thoroughly , then filter - sterilize again .
Dilute the 2x lysis buffer 1 : 1 with MQ water .
Weigh out minimum amount of ProtK , then add the appropriate amount of lysis buffer .
Filter sterilize again .
Add 1 volume of 2x Lysis buffer with Lysozyme & RNase to 1 volume of sorted cells .
Note down volumes and resulting volume V1 .
Incubate at 37°C for 30 min , rotating end - over - end at angle ( OR : in the shaking incubator @ 100 rpm , vertical ) , for optimal mixing with minimal frothing ; alternatively , shake vertically @ 100 rpm on shaking incubator and invert 10 times every 10 min .
Add V1 * 0.07 μl of ProtK lysis buffer ( 2 ) to a final concentration of 0.65 mg / ml .
Note down volumes , including resulting volume V2 .
Add V2 / 9 μl of 10 % SDS to a final concentration of 1 % .
Note down volumes .
Incubate at 55°C for 2 hours , rotating end - over - end at angle ( OR : in the shaking incubator @ 100 rpm , vertical ; invert every 20 min 10 times ) .
Add 600 μl buffer AL ( = buffer without the EtOH ) .
Mix thoroughly by vortexing .
Incubate at 70°C for 10 min ( for heat block use 1.5 ml tubes ! )
Add 600 μl of 96 - 100 % EtOH Mix by vortexing vigorously .
Check pH of lysate , must be < 7 to get max . binding efficiency .
Pipette max . possible volume onto spin columns .
Spin 3 min at 8000xg ; discard flow - through .
Pipette additional lysate onto spin columns .
Spin 1 min at 8000xg ; discard flow - through and collection tube , transfer column to new collection tube .
Add 500 μl of buffer
AW1 Spin 2 min at 8000xg ; discard flow - through .
Add 500 μl of buffer .
AW2 Spin 3 min at 8000xg ; discard flow - through and collection tube , To dry columns , spin at 8000xg for 3 min .
Discard potential flow - through ; transfer column to new collection tube .
Add 200 μl pre - heated 70°C buffer AE or water .
Incubate 1 min at room temp .
Spin at 8000xg for 2 min to elute .
Repeat with second 200 μl .
Transfer eluted DNA to the Amicon Ultra 30 K .
Centrifuge at 10,000xg for 10 min .
Rinse DNA with 500 μl PCR water .
Centrifuge at 10,000xg for 10 min .
Add 20 μl dilute TE , pipette up and down 20 times and transfer to clean tube for storage .
Centrifuge at 1000xg for 3 min upside down , in fresh tube to retrieve ( Optional : repeat with another 20 μl to ensure all retrieved )
