Apr 09, 2025
DNA Extraction with Zymo Quick-DNA™ Fungal/Bacterial Miniprep Kit
- 1University of Missouri - Veterinary Medical Diagnostic Laboratory
- University of Missouri
Protocol Citation: Zhenyu Shen 2025. DNA Extraction with Zymo Quick-DNA™ Fungal/Bacterial Miniprep Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpdw98gzp/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: April 08, 2025
Last Modified: April 09, 2025
Protocol Integer ID: 126337
Keywords: DNA extraction, bacteria, fungi, Zymo, Quick-DNA Fungal/Bacterial Miniprep Kit
Abstract
This protocol is used to extract genomic DNA from bacterial and fungal isolates using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research). The kit enables rapid DNA extraction in as little as 15 minutes. The resulting ultra-pure DNA is suitable for PCR, next-generation sequencing (NGS), and other molecular applications.
This protocol includes three key modifications to the manufacturer’s instructions:
- A sample preparation step has been added.
- Guidance is provided for addressing insufficient bead beating.
- The lysis mixture is loaded onto the column once, rather than twice, to streamline the workflow.
Materials
Kit
- Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo, D6005)
Equipment:
- Microcentrifuge
- Vortex mixer with horizontal tube holder
Supplies
- Microcentrifuge tubes
- Inoculation loops
Chemical
- β-mercaptoethanol
Before start
For a new kit, add β-mercaptoethanol (not provided) to the Genomic Lysis Buffer to a final concentration of 0.5% (v/v), i.e., 500 µL per 100 mL.
Both bacterial/fungal colonies on agar plates and broth cultures can be used for DNA extraction.
Colonies
- Colonies from fresh culture plates can be used directly. Plates can be stored at 2°C – 8°C for up to two weeks before extraction.
- Add 750 µL of BashingBead Buffer to a ZR BashingBead Lysis Tube.
- Use a sterile inoculation loop to transfer one or several pure colonies (depending on colony size) into the tube.
Note: If colonies are difficult to pick, cut a small piece of agar containing colonies and transfer the agar into the lysis tube.
Broth Culture
- Centrifuge 1 mL of culture at 6,000 × g for 10 minutes in a microcentrifuge tube. Discard the supernatant and retain the pellet. The pellet can be stored at -25°C to -15°C until DNA extraction.
- Resuspend the pellet in 750 µL of BashingBead Buffer and transfer the suspension to a ZR BashingBead Lysis Tube.
Secure the lysis tubes in a bead beater (e.g., a vortex mixer with a horizontal tube holder) and homogenize at maximum speed for 5 minutes.
Note: Do not discard the tube at this stage. If DNA extraction fails due to insufficient bead beating, you may re-process the tube by extending bead beating to 20 minutes and repeating the downstream steps.
Centrifuge the lysis tubes at 10,000 × g for 1 minute.
Transfer 200 µL of the supernatant into a Zymo-Spin‱ III-F Filter in a collection tube. Centrifuge at 10,000 × g for 1 minute.
Discard the filter. To the flow-through, add 600 µL of Genomic Lysis Buffer and mix thoroughly by pipetting.
Load the entire 800 µL mixture into a Zymo-Spin‱ IICR Column in a new collection tube. Centrifuge at 10,000 × g for 1 minute.
Place the Zymo-SpinTM IICR Column into a new collection tube. Discard the old tube with the flow-through.
Add 200 µL of DNA Pre-Wash Buffer to the column. Centrifuge at 10,000 × g for 1 minute.
Add 500 µL of g-DNA Wash Buffer to the column. Centrifuge at 10,000 × g for 1 minute.
Transfer the column to a clean 1.5 mL microcentrifuge tube (not provided). Add 35-100 µL of DNA Elution Buffer directly onto the column matrix. Centrifuge at 10,000 × g for 1 minute to elute the DNA.
Store the eluted DNA at -20°C.
Protocol references
Zymo: Manufacturer’s protocol for the Quick-DNA Fungal/Bacterial Miniprep Kit