I got to do some bona fide lab work today, which was a nice change of pace. I’ve been taking samples of larvae for DNA sequencing at various points throughout the experiment:

• From all newly released larvae (either from each family or combined, depending on how I filtered them out)
• From larvae in the “New” tanks that reach 160 microns
• From larvae in the “160” tanks that reach 224 in size
• Occasionally pooled larvae from a tank

These samples have mostly been stored at -20degC in .5-1 mL of RNALater, but duplicates of many were also stored in ethanol (1st in 75%, then in 95%). Earlier in the summer I wanted to do a test extraction to see if there was a particular storage method that worked best and figure out which extraction kit to use, but then the oysters needed maintenance 6 days a week and all of a sudden in was almost August. With the growth rate experiments and larval production essentially done, I finally had a day to do the test extraction.

I used the E.Z.N.A. Mollusc DNA Kit as recommended by the Roberts Lab. This was the 1st time I had ever used this kit, and found the instructions easy to follow but a bit more time required per sample compared to DNeasy due to a chloroform:isoamyl step and extra washes. I chose 8 larvae samples that had been taken 6/25/15-7/1/15 (right before the massive 4th of July larvae die-off). 6 of these were replicates using different storage methods.

I pretty much followed the instructions, but made a few minor modifications. In particular I tried to minimize vortex time. Protocol is below, with notes from this extraction.

1. Remove as much storage liquid as possible.
   1. Larvae stored in RNALater were floating (I thought they were somehow still swimming at first…..) so I centrifuged those samples at 10 gs for 2 minutes. They would become buoyant again quickly, and I lost some larvae from these samples trying to take out the RNALater.
2. Add 350 uL ML1 Buffer and 25 uL Proteinase K to the same tube the larvae were stored in. Use blue microtubule pestle to grind larvae at bottom of the tube. Vortex at max speed for 3 secs.
   1. Could not find enough already sterilized pestles, so wiped off with ethanol and reused a couple for storage replicates (not for different samples).
3. Incubate for 2 hours a 60degC.
   1. Checked the samples every 30 minutes- most were fully digested after an hour but 3, 4, and 8 weren’t.
4. Add 350 uL chloroform:isoamyl alcohol (24:1). Vortex at max speed for 3 secs.
5. Centrifuge 10,000 x g for 2 minutes.
6. Transfer upper aqueous phase to clean 1.5 mL tube.
7. Add one volume (500uL) MBL Buffer and 10 uL RNase A. Vortex at max speed for 8 sec.
8. Incubate at 70degC for 10 minutes. Cool to room temp.
9. Add one volume (500uL) 100% ethanol. Vortex at max speed for 8 sec.
10. Insert column into a 2mL collection tube.
11. Transfer 750 uL to column. Centrifuge at 10,000 x g for 1 minute. Discard filtrate. Repeat until all sample has passed through column.
    1. Did twice.
12. Insert Column into new collection tube. Add 500 uL HBC Buffer.
13. Centrifuge at 10,000 x g for 30 sec.Discard filtrate.
14. Add 750 uL Wash Buffer. Centrifuge at 10,000 x g for 1 min. Discard filtrate and repeat.
15. Centrifuge empty column at max speed (10,000 x g) for 2 min.
16. Transfer column to clean 1.5 mL tube.
17. Add 100 uL Elution Buffer.
    1. Did not preheat as in manual.
18. Let sit for 2 minutes. Centrifuge 10,000 x g for 1 minute.
19. Add 50 uL Elution Buffer. Repeat Step 18.
20. Store DNA at 5degC.