TITLE: Qiagen DNeasy 96 blood &amp; tissue protocol for bee guts 
AUTHORS: Lauren  Ponisio, Jocelyn Zorn

[DESCRIPTION]
Tissue protocol for bee guts. 

[STEPS]
SECTION: Day 1
1. 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 

SECTION: Day 1
2. Combine 36 mL   and 4000 µL   in 50mL centrifuge tube, invert gently to mix

SECTION: Day 1
3. Add 200 µL ATL + Proteinase K solution to each sample

SECTION: Day 1
4. Add ~100 µL zirconia beads and 2 steel beads to each sample

SECTION: Day 1
5. Place plates in tissue lyser and run at 30 Hz for 150 s, then rotate plates 180 degrees and lyse at 30 Hzfor an additional 150 s 

 

 




SECTION: Day 1
6. Centrifuge on "short" for 7 s then shake by hand for 15 s 

 

SECTION: Day 1
7. Incubate 56 °C for 60 min with weight on top of plate
Note: remove from incubator carefully with weight on top so that caps do not pop off

SECTION: Day 1
8. Place plates in tissue lyser and run at 30 Hz for 1 min 

SECTION: Day 1
9. Incubate 56 °C overnight with weight on top of plate

SECTION: Day 2
10. 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
SECTION: Day 2
11. 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
SECTION: Day 2
12. Shake plates by hand for 15 s , rotating plates halfway through

SECTION: Day 2
13. Centrifuge on "short" for 7 s  

SECTION: Day 2
14. Add 410 µL ALE to each sample

SECTION: Day 2
15. Seal with new caps and shake by hand for 15 s , rotating plates halfway through

SECTION: Day 2
16. Centrifuge on "short" for 7 s 

SECTION: Day 2
17. Place DNeasy filter plates on top of s-blocks
SECTION: Day 2
18. Transfer lysate to DNeasy filter plate and seal with AirPore tape
SECTION: Day 2
19. Centrifuge 3486 x g, 13 min . Discard flowthrough

SECTION: Day 2
20. Add 500 µL   to each sample, seal with AirPore tape

 

SECTION: Day 2
21. Centrifuge 3486 x g, 8 min . Discard flowthrough

SECTION: Day 2
22. Add 500 µL  , do not seal


 

SECTION: Day 2
23. Centrifuge 3486 x g, 20 min . Discard flowthrough

SECTION: Day 2
24. Repeat steps 22 and 23
SECTION: Day 2
25. Place DNeasy filter plate on top of elution microtube plate (dark blue)
SECTION: Day 2
26. Add 55 µL   to each sample

SECTION: Day 2
27. Incubate at room temperature for 1 min 

SECTION: Day 2
28. Centrifuge 3486 x g, 5 min 

SECTION: Day 2
29. Aliquot entirety of product into 3 96 well microplates per DNA plate, seal with labeled foil lids and store at -20 °C 

SECTION: Day 2
30. For a second backup elution, add 50 µL   to each sample
SECTION: Day 2
31. Incubate at room temperature for 1 min 

SECTION: Day 2
32. Centrifuge 3486 x g, 5 min 

SECTION: Day 2
33. Seal with white strip caps
SECTION: Day 2
34. Store at -20 °C