Mar 31, 2026
eDNA Extraction of 47mm membrane filters using Zymo Miniprep plus kit
Forked from eDNA Extraction using Zymo Miniprep plus kit
- Eldridge Wisely1
- 1University of California, San Diego
Protocol Citation: Eldridge Wisely 2026. eDNA Extraction of 47mm membrane filters using Zymo Miniprep plus kit. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dze3g1y/v1
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: March 31, 2026
Last Modified: March 31, 2026
Protocol Integer ID: 314219
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.
Materials
Zymo Miniprep plus kit
Zymo DNA/RNA Shield (optional)
5mL centrifuge tubes (sterile)
1.5mL tubes (or capless 1.5mL tubes -Axygen)
1.5mL lo-bind tubes (Axygen)
if using Qubit:
Qubit tubes, buffer and standards
Troubleshooting
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.
Extraction
add 20ul Proteinase K into all samples (in 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.
Vortex samples again 5min at RT again at speed setting 4.
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
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 new 1.5mL tubes to hold the spin columns in the next step. (or use capless 1.5mL tubes)
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.