DNEasy DNA Extraction - Vibrio Gram Negative Broth Bacteria
Put Buffer ATL and AL at 56 C for 5 minutes before using .
Transfer 250ul of broth culture to 2ml deep well ( autoclaved ) .
Harvest cells ( maximum 2 x 109 cells ) by centrifuging plate for 15 min at 4063 x g .
Discard supernatant .
Resuspend pellet in 200 µl Buffer ATL by pipetting up and down 15 times .
Seal with foil and place in oven at 56 C for one hour .
Place extra plate on seal so foil doesn’t come off .
Pipette up and down 15 times and spin down .
( May place in - 80 C and continue procedure later at this point . )
If placed in - 80 C , let thaw and spin down @ 1000g for 1 min .
Add 205 uL Buffer AL and 205 uL molecular grade EtOH .
Pipette up and down 15 times and spin down .
Place DNeasy 96 plates on top of 2ml deep well plate .
Mark the DNeasy 96 plates for later sample identification .
Carefully transfer the lysate ( approximately 600ul ) of each sample from step 7 to each well of the DNeasy 96 plates .
Do not transfer more than 900 µl per well .
Seal each DNeasy 96 plate with a porous film .
Centrifuge for 15 min at 4063g .
If lysate remains in any of the wells , centrifuge for a further 10 min .
Add 500 µl Buffer AW1 to each sample .
Seal each DNeasy 96 plate with a new AirPore Tape Sheet ( provided ) .
Centrifuge for 5 min at 4063g .
Remove the tape .
Carefully add 500 µl Buffer AW2 to each sample .
Centrifuge for 15 min at 4063g .
Do not seal the plate with AirPore Tape .
The heat generated during centrifugation ensures evaporation of residual ethanol in the sample ( from Buffer AW2 ) that might otherwise inhibit downstream reactions .
Place in new collection rack ( 2ml deep well plate ) and spin again 15 minutes at 4063g .
Place each DNeasy 96 plate in the correct orientation on a new rack of VWR 500ul plate .
To elute the DNA , add 200 µl Buffer TE to each sample , seal and incubate for 1 minute at room temp .
Centrifuge for 4 min at 4063g .
200 µl Buffer TE is sufficient to elute up to 75 % of the DNA from each well of the DNeasy 96 plate .
Recommended : For maximum DNA yield , repeat step 16 with another 200 µl Buffer TE .
Aliquot 20ul of extracted DNA to green 96 microplate .
Seal DNeasy 96 plate and 96 microplate with non - sterile foil .
Place DNeasy 96 plate in - 80 C and 96 microplate in - 20 C .
