Sep 18, 2025
Version 2
eDNA Extraction using Zymo Miniprep plus kit V.2
This  protocol is a draft, published without a DOI.
- 1University of California, San Diego
Protocol Citation: Eldridge Wisely 2025. eDNA Extraction using Zymo Miniprep plus kit. protocols.io https://dx.doi.org/Version created by Eldridge Wisely
License: This is an open access  protocol distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 18, 2025
Last Modified: September 18, 2025
Protocol Integer ID: 227632
Keywords: extracting edna, edna from sterivex filter, edna yields from this kit, extracting dna, using zymo miniprep, filtered edna, zymo miniprep, edna yield, sterivex filter, extraction, dna from many type
Abstract
The Zymo miniprep plus kit is highly versatile, and has manufacturer protocols for extracting DNA from many types of samples. In addition to the sample types mentioned in the official protocol, this kit can also be used for extracting DNA from filtered eDNA. With additional initial heating and vortexing steps, extracted eDNA yields from this kit are comparable to those from much more expensive kits. This protocol includes adaptations for extracting eDNA from Sterivex filters as well as from disk filters.
Materials
Zymo Miniprep plus kit
Zymo DNA/RNA Shield (optional)
5mL centrifuge tubes (sterile)
1.5mL tubes
1.5mL lo-bind tubes (Axygen)
if using Qubit:
Qubit tubes, buffer and standards
if extracting from Sterivex filters: 1.7mL or 2 mL micro centrifuge tubes, 50 mL centrifuge tubes, large centrifuge that can accept 50mL tubes.
Preparation
Clean extraction area with bleach or DNA Away and UV for 15 minutes minimum if possible.
turn on hula mixer/ incubator (55c at 124 rpm)
If Proteinase K is not yet prepared, add 1040ul buffer to tube of Proteinase K powder from kit.
If your samples are stored in 400uL of DNA/RNA Shield buffer (in vials, tubes, or Sterivex), add 400uL of DNA/RNA Shield to your extraction negative 5mL tube.
If samples were not stored in 400uL of DNA/RNA Shield, use either 400uL DNA/RNA Shield or 200uL of Red Biofluid and Cell buffer and 200uL of DNA Elution Buffer in all samples and also the negative control.
Sterivex only
If there is lots of sediment in your Sterivex filters, spin down in centrifuge with caps on 3000 rpm for 1 minute at room temp.
Extraction
add 20ul Proteinase K into all samples (Sterivex or 5mL tubes).
Vortex samples 5min at RT at speed setting 4
Incubate tubes at 55C for 15 min on their sides in the hula mixer 124 rpm.
Vortex samples again 5min at RT at speed setting 4
place elution buffer in incubator/hula mixer after taking out the samples.
prepare and label spin columns.
add 420ul of genomic binding buffer to each sample. pipette directly into sterivex inflow so the pipette tip touches the filter.
Vortex samples again 5min at RT again at speed setting 4.
Sterivex only
remove lysate from Sterivex: for each Sterivex filter, you will need a 50 mL centrifuge tube and a 1.7 mL or 2 mL microcentrifuge tube, and a large centrifuge that accepts 50mL tubes.
Snap off the outflow tip of the Sterivex filter and place the outflow into the microcentrifuge tube. Remove lid from inflow of Sterivex. Place both positioned together into a 50ml micro centrifuge tube.
Cap 50ml micro centrifuge tube. Centrifuge (3000 rpm for 1 minute at room temp). If all does not go through, centrifuge again.
Extraction
Transfer 840ul of lysate from samples to spin columns.
Centrifuge the spin columns at max speed (greater than or equal to 12,000x g) for 1 minute
Optional: Centrifuge the sterivex or 5mL tubes with filters in them for 5-10 seconds (short) to get more lysate. Dump liquid from collection tube into the liquid waste, then replace spin column in same collection tube.
Repeat transfer up to 840uL of lysate to spin column until all lysate has been processed.
Put spin columns in new collection tubes and then add 400uL DNA pre-wash buffer to the spin column.
Qubit
(if doing Qubit quantification after extraction, this is a good time to bring the Qubit buffer and standards to room temperature.)
Extraction
centrifuge spin columns max speed 1 minute, then empty the spin column
add 700uL of g-DNA Wash Buffer to each spin column
Centrifuge at maximum speed for 1 minute, empty the spin column again.
add 200uL of g-DNA Wash Buffer to each spin column.
Centrifuge at maximum speed for 1 minute. Cut off caps of 1.5mL tube to hold the spin columns in the next step.
Place spin columns in 1.5mL tubes with caps cut off.
Add 100 uL warm (55 C) DNA Elution Buffer to directly to the filter in the spin column.
Let sit at room temperature for 10 minutes.
Label your final 1.5mL lo-bind tubes
Centrifuge at maximum speed for 1 minute.
transfer DNA eluate a new lo-bind 1.5mL micro centrifuge tube.
Qubit
put 2 more than your number of samples of Qubit tubes into a clean empty 1000uL tip box
Add 198uL of 1x HS Qubit buffer (protect from light) to the Qubit tubes for the samples, and 190uL of 1x HS Qubit buffer to the two extra tubes.
add 2uL sample into the Qubit tubes, close and label them.
Extraction
Store sample eluate in the freezer (-20 C)
Qubit
add 10uL of each standard to the their respective tubes, close and label them.
Close tubes tightly and vortex 10 seconds, make sure there are no bubbles on the bottom. Place back in box and place box in drawer for 2 minutes minimum (but no more than 30 minutes).
plug in Qubit reader, then select the assay from the label on the Qubit buffer. (1x HS dsDNA)
run standards, using standard 1 then standard 2.
Then select run samples. Select the amount of DNA extract you're using in each tube (in this case 2 uL), and the units you want reported (usually ng/uL).
Write the concentrations in your lab notebook.