Coral DNA Extraction - Modified DNeasy PowerSoil Pro Kit
Luigi Colin
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.)
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)
Steps
Sample preparation
Gently crush a small piece of coral in a mortar and pestle (~100mg, 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 800µL of buffer CD1 and vortex briefly.
Cell lysis
Add 83µL of
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 0h 0m 10s every 0h 15m 0s (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 2h 0m 0s will results in shearing of the longer DNA fragments
Inhibitor's removals
Add 200µL buffer CD2 and vortex for0h 0m 5s
Microfuge a 16000x g .
Transfer supernatant to new tube (Max 700µL).
Bind DNA
Add600µL of buffer CD3, vortex for 0h 0m 5s
Load 650µL of the lysate onto an MB Spin Column and centrifuge at 15000x g
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
Add 500µL of Solution EA to the MB Spin Column. Centrifuge at 15000x g
Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
Add500µL of Solution C5 to the MB Spin Column. Centrifuge at 15000x g
Discard the flow-through and place the MB Spin Column into a new 2 ml Collection
Centrifuge at up to 16000x g. Carefully place the MB Spin Column into a new 1.5ml Elution Tube.
Elute
Add 50µL–100µL of Solution C6 to the centre of the white filter membrane.
Note: To improve the yield use the lower volume and add a 0h 10m 0s incubation at room temperature
Centrifuge at 15000x g. Discard the MB Spin Column. The DNA is now ready for downstream applications.
