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DNA_Extraction.md

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DNA extraction for coral

This protocol uses the Omega Bio-Tek E.Z.N.A. Soil kit. This is the link:

https://www.omegabiotek.com/product/soil-dna-extraction-kit-e-z-n-a-soil-dna-kit/?cn-reloaded=1#protocols

Otherwise, follow these steps for coral DNA extraction.

Preparation

  • Heat a heating block/incubator to 70 ◦C, no shaking, put Elution buffer in
  • Heat another incubator to 56 ˚C, light shaking
  • Make sure your P2 buffer is chilled
  • Crosslink 3 x # of samples in 1.5 mL Eppendorf tubes
  • Make sure HBC buffer and wash buffer are correctly diluted with appropriate chemicals

Tissue lysis

  1. Weigh approximately 150 – 250 mg of coral sample in an Eppendorf tube or directly into the disruptor tube. Weigh your sample and note down how much sample you’ve processed.

The disruptor tube is faster, but you cannot put the sample back if you get too much sample.

  1. Add 725 µL of SLX-Mlus buffer to the disruptor tube. Vortex until the coral skeleton is virtually bare (usually 5-10 minutes).

  2. Centrifuge shortly to remove droplets from the lid.

  3. Add 72 µL DS buffer to each sample. Incubate at 70 degrees for 5 minutes.

  4. Vortex samples briefly.

  5. Incubate for another 5 minutes at 70 degrees.

  6. Add 3 µL RNAse and incubate for 2-3 minutes at 37 ˚C.

  7. Centrifuge at 10000 g/rpm for 5 minutes at room temperature.

Inhibitor Removal

  1. Transfer 400 µL supernatant into a 1.5 mL Eppendorf tube. Add 135 µL chilled P2 buffer and 200 µL of resuspended cHTR reagent.

  2. Centrifuge at 10000 g/rpm for 1 minute at room temperature.

Do not shake the cHTR reagent bottle: this foams it up and barely does anything for the resuspension. Its better to just pipette up and down.

Binding

  1. Transfer supernatant to a new centrifuge tube, make sure not to disturb the pellet. Add an equal volume of X1 buffer (about 735 µL)

  2. Insert a HiBind column into a 2 mL collection tube. Transfer 700 µL of the sample into the column. Centrifuge at 10000 g/rpm for 1 minute at room temperature. Discard the filtrate and reuse the collection tube.

  3. Repeat step 11 until all filtrate has passed through.

Washing

  1. Add 500 µL of HBC buffer to the column. Centrifuge at 10000 rpm/g for 1 minute at room temperature. Discard filtrate.

  2. Add 700 µL DNA wash buffer to the column. Centrifuge at 10000 rpm/g for 1 minute at room temperature. Discard filtrate.

  3. Repeat step 14.

  4. Centrifuge the column without loading it with anything for 2 minutes at maximum speed at room temperature.

Elution

  1. Transfer the column to a clean empty microcentrifuge tube. Add 50 µL elution buffer, heated to 70 ˚C. Incubate for 10 minutes at room temperature. Centrifuge at maximum speed for 1 minute.

  2. Keep the column in the same microcentrifuge tube and add 25 µL elution buffer, heated to 70 ˚C. Incubate at room temperature for 10 minutes. Centrifuge at maximum speed for 1 minute.

  3. Measure concentration and store DNA at -20 ˚C