Soaked mortar, pestle, metal spatulas in 10% bleach/DI water (100mL Clorox bleach, 900mL DI water) for ~15-20 minutes. Rinsed with DI water, let dry then covered individually in foil and autoclaved at 121C for 20 minutes.

Next day, put mortar & pestles in -20 for a copule hours. Picked up 6L of liquid nitrogen - note this is availabe in the Bagley, the Chem supply store, not in the J-wing of the microbiology building where we get dry ice.

Labeled and weighed 1.5mL microcentrifuge tubes. Got larvae and mantle tissue out of the -80 for my test run; Held samples on wet ice until homogenized.

Used E.Z.N.A. Mollusc DNA Kit. Followed kit instructions, then quantified using QBit Assay kit. A couple notes:

• Pulverized tissues in ceramic mortar/pestles in liquid nitrogen (with some difficulty … see below).
• We have 8 mortar/pestle sets in our lab. Can’t do more than 8 samples / day. * Incubated with proteinase K for 2 hours
• The “one volume” used for steps 7 & 10 was 450uL (eyeballed vol. of supernatant pulled off in step 6, and matched that).
• I did not do optional step to equilibrate columns (via NaOH)
• In step 27, for the 2 rinses I used 100uL Elution Buffer, for total volume of 200umL in each sample
• Samples are stored in the -20 in FTR 209, on door.

For larvae, they were all at the bottom of the tube. I inverted the tube and tapped vigorously to get as much onto the mortar as possible; also used autoclaved toothpicks to try to get more out. Probably shouldn’t do this next time - find another small item to scrape tissue out of tube. Curious if I can add ethanol or something to get the larvae into solution, then pour into mortar? Will ask the lab.

Poured LN2 into mortar with pestle also in; grouned tissue down. Used metal spatula to scrape frozen tissue dust into pile, carefully transferred into new 1.5mL microcentrifuge tube. This was also very difficult, as the spatula just barely fit into the tube, and if the tissue started to melt at all it would stick to the spatula. Need a better tool for transferring tissue - smaller spatula with round tip? Also, sterilized the spatula by soaking in bleach, then 2 DI rinses, between every-other sample (used 2 spatulas). Adequate? Will ask lab.

Need no more than 30mg for each sample; weighed tubes+samples. For samples with >30mg, labeled and weighed new tubes and either transferred ~30mg into newly labeled tube or removed a bit of sample.

Added 350uL buffer, and 25uL proteinase K. Floated in 60C water bath. Kit says to incubate for 30 mins - 4 hours; I incubated for 2 hours.

Quantified using the Qubit dsDNA HS Assay Kit, pipetting 10uL of each sample for the assay. Samples # 11, 13 & 16 were too concentrated - out of range - so I added 50uL more Elution Buffer, vortexed, and re-analyzed. Adjusted concentrations by x1.25. Better results this time! Checking with Sam & Steven to see if my concentrations meet the sequence facility’s min. DNA requirements?:

DNA VIAL #	SAMPLE #	Treatment	TISSUE TYPE	tissue mass (ug)	DNA Concentration (ng/uL)	Total ng DNA after assay	1% of DNA (ng)
test10-B	27-A	HL-10-Low	Frozen larvae	35	10	1,900	19
test11-B	60-A	HL-10-Low	Frozen larvae	36.9	12.1	2,299	23
test12	73-A	HL-10-Low	Frozen larvae	12.6	5.5	1,045	10
test13	59-A	HL-6-Ambient	Frozen larvae	29.5	12.75	2,423	24
test14	HL-10-12	HL-10-Low	Frozen mantle	28.1	11.4	2,166	22
test15	HL-10-10	HL-10-Low	Frozen mantle	14.1	6.18	1,174	12
test16	HL-6-18	HL-6-Ambient	Frozen mantle	34.9	10.475	1,990	20
test17	HL-6-20	HL-6-Ambient	Frozen mantle	34.5	4.68	889	9

I will plan on processing my “actual” samples on Monday.

To summarize issues I encountered tonight:

• Getting larvae out of sample tube. they are at the bottom of the tube, and semi-liquid once thawed. I inverted the tube and tapped vigorously into the mortar, but I leave a bunch of larvae behind in the tube. I tested adding liquid nitrogen directly into the microcentrifuge tube, which works OK. Could try using disposable pipette, with tip cut off. Checking to see if anyone has found the best method.
• Transferring powdered/frozen sample into clean tube. Metal spatulas we have are too big. I ordered small metal spatulas / scoops, arriving Sunday - ordered 9.
• Sterilizing metal spatulas used to scrape/transfer frozen tissue. Not sure if it’s adequate to soak metal spatulas in 10% bleach, then rinse twice with DI water, between samples. Asked the lab.