Nov 03, 2022
Qiagen DNeasy 96 blood & tissue protocol for bee guts
- 1University of Oregon
- FFAR
- Ponisiolab
Protocol Citation: Lauren Ponisio, Jocelyn Zorn 2022. Qiagen DNeasy 96 blood & tissue protocol for bee guts . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g78k23lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2021
Last Modified: November 03, 2022
Protocol Integer ID: 52931
Keywords: tissue protocol for bee gut, bee gut, qiagen dneasy 96 blood, tissue protocol, qiagen dneasy, blood, tissue
Funders Acknowledgements:
Foundation of Food and Agriculture Research
Grant ID: CA18-SS-0000000009
Abstract
Tissue protocol for bee guts.
Protocol materials
Proteinase K (2 ml)QiagenCatalog #19131
Buffer ATL (tissue lysis buffer)QiagenCatalog #19076
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
Day 1
1h 6m 22s
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 36 mL Buffer ATL (tissue lysis buffer)QiagenCatalog #19076 and 4000 µL Proteinase K (2 ml)QiagenCatalog #19131 in 50mL centrifuge tube, invert gently to mix
Add 200 µL ATL + Proteinase K solution to each sample
Add ~100 µL zirconia beads and 2 steel beads to each sample
Place plates in tissue lyser and run at 30 Hz for 00:02:30 , then rotate plates 180 degrees and lyse at 30 Hz for an additional 00:02:30
Equipment
TissueLyser II
NAME
Bead Mill
TYPE
QIAGEN
BRAND
85300
SKU
LINK
Equipment
TissueLyser Adapter Set 2 x 96
NAME
Adapter set
TYPE
Qiagen
BRAND
69984
SKU
LINK
5m
Centrifuge on "short" for 00:00:07 then shake by hand for 00:00:15
Equipment
Eppendorf™ 5810R Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
02-262-8187
SKU
LINK
22s
Incubate 56 °C for 01:00:00 with weight on top of plate
Note: remove from incubator carefully with weight on top so that caps do not pop off
1h
Place plates in tissue lyser and run at 30 Hz for 00:01:00
1m
Incubate 56 °C overnight with weight on top of plate
Day 2
53m 44s
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 00:00:15 , rotating plates halfway through
15s
Centrifuge on "short" for 00:00:07
7s
Add 410 µL ALE to each sample
Seal with new caps and shake by hand for 00:00:15 , rotating plates halfway through
15s
Centrifuge on "short" for 00:00:07
7s
Place DNeasy filter plates on top of s-blocks
Transfer lysate to DNeasy filter plate and seal with AirPore tape
Centrifuge 3486 x g, 00:13:00 . Discard flowthrough
13m
Add 500 µL Buffer AW1QiagenCatalog #19081 to each sample, seal with AirPore tape
Safety information
Buffer AW1 contains guanidine salt - do not mix with bleach
Centrifuge 3486 x g, 00:08:00 . Discard flowthrough
8m
Add 500 µL Buffer AW2QiagenCatalog #19072 , do not seal
Safety information
Manage and dispose of buffer AW2 as hazardous waste
Centrifuge 3486 x g, 00:20:00 . Discard flowthrough
20m
Repeat steps 22 and 23
Place DNeasy filter plate on top of elution microtube plate (dark blue)
Add 55 µL Buffer AEQiagenCatalog #19077 to each sample
Incubate at room temperature for 00:01:00
1m
Centrifuge 3486 x g, 00:05:00
5m
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 50 µL Buffer AEQiagenCatalog #19077 to each sample
Incubate at room temperature for 00:01:00
1m
Centrifuge 3486 x g, 00:05:00
5m
Seal with white strip caps
Store at -20 °C