Qiagen DNeasy 96 blood & tissue protocol for bee guts

UV sterilize supplies for 2 96 well plates worth of extractions: 1 50mL centrifuge tube, 1 15mL centrifuge tube, 1 box 1,000uL pipette tips, 2 boxes 200uL pipette tips, 24 elution microtube strip caps, steel beads, zirconia beads, and 1 reagent reservoir

Combine 36mL and 4000µL in 50mL centrifuge tube, invert gently to mix

Add 200µL ATL + Proteinase K solution to each sample

Add ~100µL zirconia beads and 2 steel beads to each sample

Place plates in tissue lyser and run at 30Hz for 0h 2m 30s, then rotate plates 180 degrees and lyse at 30Hzfor an additional 0h 2m 30s

Equipment

Value	Label
TissueLyser II	NAME
Bead Mill	TYPE
QIAGEN	BRAND
85300	SKU

Equipment

Value	Label
TissueLyser Adapter Set 2 x 96	NAME
Adapter set	TYPE
Qiagen	BRAND
69984	SKU

Centrifuge on "short" for 0h 0m 7s then shake by hand for 0h 0m 15s

Equipment

Value	Label
Eppendorf™ 5810R Centrifuge	NAME
Centrifuge	TYPE
Eppendorf	BRAND
02-262-8187	SKU

Incubate 56°C for 1h 0m 0s with weight on top of plate

Note: remove from incubator carefully with weight on top so that caps do not pop off

Place plates in tissue lyser and run at 30Hz for 0h 1m 0s

Incubate 56°C overnight with weight on top of plate

UV sterilize supplies: 4 reagent reservoirs, 24 elution microtube strip caps (clear), 2 boxes 1,000uL tips (plus some extra), 6 boxes 200uL tips (plus some extra), 2 s-blocks, 2 elution microtube plates (dark blue) and 24 strip caps (white), and 6 96 well microplates (for aliquotting final product). Label 6 foil microplate lids for aliquots

Carefully remove plates from incubator, leaving the weight on top, and allow to sit at room temperature for a few minutes before carefully removing weight to ensure strip caps do not pop off

Shake plates by hand for 0h 0m 15s , rotating plates halfway through

Centrifuge on "short" for 0h 0m 7s

Add 410µL ALE to each sample

Seal with new caps and shake by hand for 0h 0m 15s , rotating plates halfway through

Centrifuge on "short" for 0h 0m 7s

Place DNeasy filter plates on top of s-blocks

Transfer lysate to DNeasy filter plate and seal with AirPore tape

Centrifuge 3486x g . Discard flowthrough

Add 500µL to each sample, seal with AirPore tape

Centrifuge 3486x g . Discard flowthrough

Add 500µL , do not seal

Centrifuge 3486x g . Discard flowthrough

Repeat steps 22 and 23

Place DNeasy filter plate on top of elution microtube plate (dark blue)

Add 55µL to each sample

Incubate at room temperature for 0h 1m 0s

Centrifuge 3486x g

Aliquot entirety of product into 3 96 well microplates per DNA plate, seal with labeled foil lids and store at -20°C

For a second backup elution, add 50µL to each sample

Incubate at room temperature for 0h 1m 0s

Centrifuge 3486x g

Seal with white strip caps

Store at -20°C