Notes on DNA extractions of the second batch of 0.45um 1/2 water filters from FHL 2026 experiment.

background info

Experiment info: Lab Notebook Post

DNA extraction info

Samples

–> extracted DNA from n=23 0.45um water filters (cut in half –> remaining half of filter is in FTR -80C) and 1 blank sample (DNA extraction control).

Samples extracted:

sample_id treatment_id treatment sample_type vol_filtered_mL sample_location extraction_batch DNAextract_date amount_sample_processed extraction_kit DNA_location elution_liquid elution_vol_ul notes
F_T0_01 NA 0.22um filtered SW + Vpec 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_ESW_04 ESW eelgrass + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_ESW_06 ESW eelgrass + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_ESW_08 ESW eelgrass + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EVP_03 EVP eelgrass + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EVP_08 EVP eelgrass + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_VPSW_03 VPSW vp + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_VPSW_07 VPSW vp + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EM_01 EM eelgrass + mussel + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EM_02 EM eelgrass + mussel + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EM_04 EM eelgrass + mussel + sw 0.45um filter 35 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 May 27th 11:33pm At bagging time - mussel was spawning (male); half filter remaining in -80C; DNA from half filter
F_EM_05 EM eelgrass + mussel + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EMVP_01 EMVP eelgrass + mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EMVP_04 EMVP eelgrass + mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EMVP_05 EMVP eelgrass + mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_EMVP_08 EMVP eelgrass + mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_MVP_02 MVP mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_MVP_08 MVP mussel + vp 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_ShSW_04 ShSW mussel shell + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_ShSW_08 ShSW mussel shell + sw 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_SW_03 SW seawater + media 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_SW_04 SW seawater + media 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
F_SW_07 SW seawater + media 0.45um filter 150 -80 FHL 2 2026-07-07 half-filter ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50 half filter remaining in -80C; DNA from half filter
BLANK2 BLANK BLANK BLANK NA NA 2 2026-07-07 NA ZymoBIOMICS DNA Miniprep Kit (D4300) FTR -80C water 50  

Kit and protocol info:

ZymoBIOMICS DNA MiniPrep Kit –> D4300

Extraction protocol notes: Protocol_eDNA-extraction_protocol_GAC_2026Samples

I’m now using a centrifuge that can hold 24 tubes - though the 24-tube centrifuge that was in FTR 213 has an error code (error 3), so I walked back and forth between 213 and 209 (used the 24-tube centrifuge in 209) to do the protocol.

During the near-to-last step (Step 11, Page 7), I put 50ul of water into the first sample (F-T0_01) to elute DNA and went to close the cap, and my finger slipped and all the tubes went flying. So there is a chance of cross-contamination, however none of the tubes except that one sample had any liquid in it and the DNA was trapped on the filter. The F-T0-01 tube fell on the ground (cap was open), while all the others were on the benchtop. I proceeded and if the qPCR looks weird for those samples, I’ll re-extract from the other half of the filter in the -80C. As a note: since I wasn’t sure if liquid was in the filter for F-T0-01, I added another 50ul so the elution volume is >50ul, will need to measure before I run 2ul on qPCR for downstream calculations. Photo below:

img

Up next

  1. Finish extracting DNA from remaining water filters (n=35 remaining)
  2. Run 2ul of each sample’s DNA on qPCR looking for V. pectenicida <– late July (out of town July 10-20).

NOTE!!!

The TaqMan Fast Universal PCR Master Mix arrived 1+ week ago and I never was alerted… I asked about the order today because I hadn’t received any tracking or delivery info, and was told that it was delivered, so then I went to SAFS front desk and asked, and we looked in the freezer storage and found it. A drafted email that was supposed to be sent to me telling me it arrived was never sent - so it’s important to check on shipments because you never know if it was sent or delivered! I thought it seemed like too long, which is why I asked about it today. The TaqMan was still cold and is meant to be stored at 4C.

I called customer service to get thoughts on it and here are the notes from that call:

  • Received that order was completed 6/29/2026, but no tracking info

  • Checked today - emailed Janna Southworth asking if it was ordered and if there was tracking info

  • She said it was delivered → sent me invoice that it was delivered to central receiving 6/26/2026

  • It was sent to UW SAFS, but no email to me

  • Johnny found that it was received and that an email was drafted to send me June 30th, but it wasn’t sent

  • Called customer service to ask if the TaqMan is ok to use:

Stored at freezer for 1 week
Doesn’t have any stability data on the freezer
Usually with freeze thaws on the enzymes → not sure if this one would totally freeze

Look: If froze completely, enzyme would be in ice crystals and could destroy the master mix <– tubes were liquid
Likely not a concern
If see any precipitate → don’t want those
Vortex it before use and see if see anything coming out of solution and that would be where we’d be concerned
No data to back up if it works fine

Make sure mix is well mixed before use!!!!