Notes on protocol and process in post. Testing again with correct mussel tissue weights.

Why am I doing this again?

Well… it turns out I messed up the decimals when I was weighing my tissue during the first FHL 2025 mussel tissue DNA extraction test, and was testing out 100mg, 150mg, 200mg, and 250mg of tissue instead of 10mg, 15mg, 20mg, and 25mg. So I am re-doing the test with the correct weights.

GitHub Issue with response from SR: #2507

Kit and Protocol Notes:

Extraction performed 2026-10-13

Protocol notes: here

The kit reagents that I got from Olivia Graham is missing extra Proteinase K, so a previous notebook post explains that I can use the proteinase K from old Zymo kits, so long as it’s in the correct concentration (20mg/mL).

I looked back at the Zymo Quick DNA/RNA Microprep Plus Kit (D7005) protocol and saw that the aliquots I made for that kit are in the correct concentration (20mg/mL). So I am using those moving forward. I have a bunch aliquoted out! Stored in the FTR 213 freezer.

Samples Extracted

I grabbed 4 mussels from the 2025 FHL pilot experiment that were not actually from the experiment at all… but were exposed to Vibrio pectenicida for a couple days.

The samples are:

M2A; M2B; M3A; M3B

To sample the tissue, for each sample, I dissected out the whole mussel body and placed into a clean 1.5ml tube. I then used a dissection tool (pictured below) to mash up the tissue in the tube… I then used sterile tweezers and scissors to measure out the desired weights… is there a better way to do this?? It’s such small tissue weight. See pictures below.

dissecting tool i used to mash up the tissue:

each mussel tissue sample looked like this (whole body) after mashing it up with the tool:

each tissue subsample weighed to the correct weights were reallllyyyyy tiny samples, like this one:

Qubit and Nanodrop results:

Qubit

Using the Invitrogen Qubit 1X dsDNA HS Kit protocol.

Letting the reagents thaw to room temp for 30 mins. Letting the DNA thaw to room temp as well.

Running 2ul of each sample, so the math is:

Standard 1 –> 10ul of standard 1 + 190ul of working solution
Standard 2 –> 10ul of standard 2 + 190ul of working solution
Sample 2A –> 2ul of sample + 198ul of working solution
Sample 2B –> 2ul of sample + 198ul of working solution
Sample 3A –> 2ul of sample + 198ul of working solution
Sample 3B –> 2ul of sample + 198ul of working solution

Re-ran 3A and 3B with new standards with 1ul of sample:
Standard 1 –> 10ul of standard 1 + 190ul of working solution
Standard 2 –> 10ul of standard 2 + 190ul of working solution
Sample 3A –> 1ul of sample + 199ul of working solution
Sample 3B –> 1ul of sample + 199ul of working solution

Nanodrop

Ran 2ul of each sample on the nanodrop.

Goal is to have “Nanodrop is still useful for the purity ratios. Look at 260/280 (target about 1.8) and 260/230 (target about 2.0 to 2.2). A low 260/230 would point to carryover”

All Results:

“Look at 260/280 (target about 1.8) and 260/230 (target about 2.0 to 2.2). A low 260/230 would point to carryover”

None of these fit the targets for the nanodrop values….

For my actual samples:

I have n=32 samples from the actual FHL 2026 Experiment (experiment info here).