Extracting DNA from C. virginica sperm samples

We got C. virginica sperm and mantle samples from the Lotterhos Lab’s 2018 experiment! I’m going to use sperm samples to examine potential for epigenetic inheritance in this species. But first, I need to see if I can get usable DNA from these samples.

Sam suggested I use the E.Z.N.A Mollusc Kit for these extractions instead of DNAzol like Claire did with her sperm samples. I’m going to test this method on two sperm samples: L18A0012s and L18A0031s.

Methods

Step 1. Obtain liquid nitrogen (LN2), dry ice, a small ceramic mortar and pestle, and a wide spatula. Place DNA samples in dry ice. Set a heat block to 37ºC.

Step 2. Pour LN2 into the ceramic mortar, and additional LN2 into a styrofoam cooler. Place the pestle and spatula in the cooler. While waiting for the LN2 to boil off the mortar, prepare a 10% bleach solution.

Step 3. Once the LN2 has boiled off, transfer the DNA sample (no more than 30 mg) into the mortar. Break the frozen sample with the spatula, then transfer into the mortar. If the sample does not break, slightly thaw it with heat from your hand.

Step 4. Pulverize the DNA sample with the LN2-cooled pestle. Transfer the powder to a clean, labeled 1.5 mL microcentrifuge tube.

Step 5. Obtain the E.Z.N.A. Mollusc Kit. Add 350 µL ML1 Buffer and 25 µL Proteinase K Solution to each sample. Vortex thoroughly.

Step 6. Place the samples on 37ºC heat block for overnight incubation.

Going forward

1. Wednesday: Finish isolating DNA and quantify with the Qubit. If successful, pulverize remaining samples and start overnight incubation
2. Thursday: Finish isolating DNA from all samples and quantify with the Qubit