Oct 27, 2021

Public workspaceCoral DNA Extraction - Modified DNeasy PowerSoil Pro Kit

DOI
dx.doi.org/10.17504/protocols.io.bww6pfhe
  • 1Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK
Luigi Colin
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DOI: dx.doi.org/10.17504/protocols.io.bww6pfhe
External link: https://doi.org/10.3897/BDJ.9.e72762
Protocol Citation: Luigi Colin 2021. Coral DNA Extraction - Modified DNeasy PowerSoil Pro Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bww6pfhe
Manuscript citation:
Colin L, Yesson C, Head CEI, Complete mitochondrial genomes of three reef forming corals (, ) from Chagos Archipelago, Indian Ocean. Biodiversity Data Journal doi: 10.3897/BDJ.9.e72762
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
Created: July 27, 2021
Last Modified: October 27, 2021
Protocol Integer ID: 51902
Keywords: DNA extraction, Acropora, Scleractinian, Qiagen, coral, DNA
Funders Acknowledgements:
Bertarelli Programme in Marine Science
Abstract
DNeasy PowerSoil Pro Kit modified extraction protocol to improve yield with inhibitor heavy hard corals.
Tested and developed on Acropora corals. Optimised for all coral DNA extraction that with high quantity of inhibitors

(Step 1 to 8 modified protocol; step 9 to 18 manufacturer’s protocol.)
Materials
  • DNeasy PowerSoil Pro Kit
  • Proteinase K
  • Mortar and pestle
  • Sharp scalpel
  • Microcentrifuge (up to16,000 xg)
  • Pipettor (50–1000 μl)
  • Vortex-Genie2
Protocol materials
ReagentProteinase K (2 ml)QiagenCatalog #19131
Before start
  • Sterilise all the equipment that is not single use.
  • Ensure that the sample are as dry as possible before starting the protocol, taking particular care of removing any buffer they may have been kept in (Especially if RNALater was used)
Sample preparation
Sample preparation
2h 33m 20s
2h 33m 20s
Gently crush a small piece of coral in a mortar and pestle (~Amount100 mg , in order not to overload the column though). And transfer in a PowerBead Pro Tube (or 2 ml Microcentrifuge).
Note: For improved yield focus on adding more tissue and less carbonate from the skeleton, a scalpel
Add Amount800 µL of buffer CD1 and vortex briefly.

Cell lysis
Cell lysis
2h 33m 20s
2h 33m 20s
Add Amount83 µL of ReagentProteinase K (2 ml)QiagenCatalog #19131 and vortex briefly.
Note: quality of Proteinase K can be tweaked depending on the quality and type of samples
Incubate at 56 degrees for 60 minutes. Vortex for Duration00:00:10 every Duration00:15:00 (Ideally, do this on a shaking heat block, with shaking on full)

Note: If on orbital incubator at a minimum of 300rpm or more, can skip the vortexing.
Add an extra hour of incubation to increase yield. More than Duration02:00:00 will results in shearing of the longer DNA fragments

2h 15m 10s
Inhibitor's removals
Inhibitor's removals
2h 33m 20s
2h 33m 20s
Add Amount200 µL buffer CD2 and vortex forDuration00:00:05

5s
Microfuge a Centrifigation16000 x g, Room temperature, 00:01:00 .

1m
Transfer supernatant to new tube (Max Amount700 µL ).
Expected result
Expect ~ Amount650 µL



Bind DNA
Bind DNA
2h 33m 20s
2h 33m 20s
AddAmount600 µL of buffer CD3, vortex for Duration00:00:05

5s
Load Amount650 µL of the lysate onto an MB Spin Column and centrifuge at Centrifigation15000 x g, Room temperature, 00:01:00

1m
Discard the flow-through and repeat step 9 to ensure that all the lysate has passed through the MB Spin Column.
Carefully place the MB Spin Column into a clean 2 ml Collection Tube. Avoid splashing any flow-through onto the MB Spin Column.

Wash
Wash
2h 33m 20s
2h 33m 20s
Add Amount500 µL of Solution EA to the MB Spin Column. Centrifuge at Centrifigation15000 x g, Room temperature, 00:01:00

1m
Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
AddAmount500 µL of Solution C5 to the MB Spin Column. Centrifuge at Centrifigation15000 x g, Room temperature, 00:01:00

1m
Discard the flow-through and place the MB Spin Column into a new 2 ml Collection
Centrifuge at up to Centrifigation16000 x g, Room temperature, 00:03:00 . Carefully place the MB Spin Column into a new 1.5ml Elution Tube.

3m
Elute
Elute
2h 33m 20s
2h 33m 20s
Add Amount50 µL –Amount100 µL of Solution C6 to the centre of the white filter membrane.

Note: To improve the yield use the lower volume and add a Duration00:10:00 incubation at room temperature

10m
Centrifuge at Centrifigation15000 x g, Room temperature, 00:01:00 . Discard the MB Spin Column. The DNA is now ready for downstream applications.

1m