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.
|1||South Sound||SS1,SS4||100||6/25/2015||75/95 EtOH||400|
|3||South Sound||SS3,SS4 (all)||100||7/1/2015||RNALater||1200|
|4||South Sound||SS3,SS4 (all)||100||7/1/2015||75/95 EtOH||1200|
|6||Hood Canal||HC1.HC3,HC4||100||6/25/2015||75/95 EtOH||400|
|7||Fidalgo Bay||NF5||100||6/24/2015||75/95 EtOH||460|
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.
- Remove as much storage liquid as possible.
- 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.
- 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.
- Could not find enough already sterilized pestles, so wiped off with ethanol and reused a couple for storage replicates (not for different samples).
- Incubate for 2 hours a 60degC.
- Checked the samples every 30 minutes- most were fully digested after an hour but 3, 4, and 8 weren’t.
- Add 350 uL chloroform:isoamyl alcohol (24:1). Vortex at max speed for 3 secs.
- Centrifuge 10,000 x g for 2 minutes.
- Transfer upper aqueous phase to clean 1.5 mL tube.
- Add one volume (500uL) MBL Buffer and 10 uL RNase A. Vortex at max speed for 8 sec.
- Incubate at 70degC for 10 minutes. Cool to room temp.
- Add one volume (500uL) 100% ethanol. Vortex at max speed for 8 sec.
- Insert column into a 2mL collection tube.
- Transfer 750 uL to column. Centrifuge at 10,000 x g for 1 minute. Discard filtrate. Repeat until all sample has passed through column.
- Did twice.
- Insert Column into new collection tube. Add 500 uL HBC Buffer.
- Centrifuge at 10,000 x g for 30 sec.Discard filtrate.
- Add 750 uL Wash Buffer. Centrifuge at 10,000 x g for 1 min. Discard filtrate and repeat.
- Centrifuge empty column at max speed (10,000 x g) for 2 min.
- Transfer column to clean 1.5 mL tube.
- Add 100 uL Elution Buffer.
- Did not preheat as in manual.
- Let sit for 2 minutes. Centrifuge 10,000 x g for 1 minute.
- Add 50 uL Elution Buffer. Repeat Step 18.
- Store DNA at 5degC.