Apr 19, 2023

Public workspaceThe extraction of cross-kingdom biofilm DNA using Zymo Reasearch Quick-DNA Fecal/Soil Microbe Kits. V.1

This protocol is a draft, published without a DOI.
  • 1UK Centre for Ecology & Hydrology;
  • 2UK Centre for Ecology & Hydrology (UKCEH);
  • 3Environment Agency
Lindsay Kate Newbold
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Protocol Citation: Lindsay Kate Newbold, Daniel S Read, Joe Taylor, Kerry Walsh, Jonathan Warren 2023. The extraction of cross-kingdom biofilm DNA using Zymo Reasearch Quick-DNA Fecal/Soil Microbe Kits.. protocols.io https://protocols.io/view/the-extraction-of-cross-kingdom-biofilm-dna-using-crzmv746
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 29, 2023
Last Modified: April 19, 2023
Protocol Integer ID: 79629
Funders Acknowledgement:
Environment Agency
Grant ID: Optimisation of biofilm DNA extraction method (SC220013)
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Abstract
The extraction of DNA from environmental samples, including soils, sediments, water, and biofilms, is a crucial stage in the analysis of environmental microbial communities and and wider environmental communities via environmental DNA (eDNA). The choice of DNA extraction methodologies significantly influences not only the yield and purity of the extracted DNA, but also the composition of the community as determined by DNA analysis techniques such as amplicon and metagenomic sequencing. Numerous studies have investigated the influence of different kit-based DNA extraction protocols on metabarcoding of various taxa, including bacteria, fungi, diatoms, microeukaryotes, and invertebrates (Dopheide et al. 2019; Giangacomo et al. 2021; Matsuoka et al. 2022; Shaffer et al. 2022; Vasselon et al. 2017; Pearman et al. 2020; Majaneva et al. 2018; Deiner et al. 2015; Djurhuus et al. 2017; Kawato et al. 2021). Here we present an amended version of the manufacturers (Zymo research) recommneded protocol for Quick-DNA Fecal/Soil Microbe Kits optimised for the extraction of cross-kingdom biofilm community DNA suitable for downstream applications such as metabarcoding.
Guidelines
This protocol is based upon the recommended manufacturers protocol for Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit (D6012) and Quick-DNA Fecal/Soil Microbe 96 Kit (D6011). When using the 96 Kit volumes should be adjusted as per manufacturers recomendations.
Safety warnings
Please read material SDS sheet before starting protocol.Download _d6011_quick-dna_fecalsoil_microbe_96_kit.pdf_d6011_quick-dna_fecalsoil_microbe_96_kit.pdf

Before start
Add Amount500 µL beta-mercaptoethanol per 100ml Genomic Lysis Buffer.

Sample Preparation
Sample Preparation
15m
15m
In field samples are taken by the Environment Agency, and Amount5 mL biofilm suspension preserved in equal amounts of preservation buffer: Concentration3.5 Molarity (M) ammonium sulphate, Concentration17 millimolar (mM) sodium citrate, Concentration13 millimolar (mM) EDTA. Samples are frozen in
Equipment
Centrifuge tube
NAME
15 ml Centrifuge tube
TYPE
Corning
BRAND
CLS430791-500EA
SKU
https://www.sigmaaldrich.com/GB/en/product/sigma/cls430791
LINK
prior to laboratory preparation.

Once back in the laboratory field preserved samples are first thawed and then vortexed to create a homogenous mixture.
Samples are pelleted at Centrifigation3000 x g, 5°C for Duration00:15:00 minutes

15m
Ensure samples are fully pelleted, then remove Amount500 µL of buffer to separate microcentrifuge tube.
Equipment
2 ml Eppendorf Safe-Lock tube
NAME
microcentrifuge tube
TYPE
Eppendorf
BRAND
0030120094
SKU
https://www.eppendorf.com/gb-en/eShop-Products/Laboratory-Consumables/Tubes/Eppendorf-Safe-Lock-Tubes-p-0030120094
LINK
Remaining buffer is gently aspirated to avoid disrupting pellet.

Pellet is resuspended in Amount500 µL of reserved preservation buffer, and stored in the same fully labelled microcentrifuge tube. Preserved sample is then frozen at Temperature-80 °C prior to analysis.

Sample Lysis
Sample Lysis
1h 11m
1h 11m
Thaw sample to room temperature, vortex to resuspend pellet and add Amount100 µL to ZR BashingBead Lysis tube. Add Amount900 µL DNA/RNA shield, and lyse for Duration00:40:00 on a horizontal vortex mixer (Vortex Genie).

40m
Briefly spin for Centrifigation10000 x g, 18°C, 00:01:00 . Add Amount40 µL proteinase K, resuspend and mix through pipetting and incubate at Temperature65 °C for Duration00:20:00 .

21m
Centrifuge ZR BashingBead tube at Centrifigation10000 x g, 18°C, 00:05:00 to pellet lysate.

5m
Transfer up to Amount400 µL supernatant to a Zymo-Spin III-F Filter in a collection tube and centrifuge at Centrifigation8000 x g, 18°C, 00:01:00 .

1m
Add Amount1200 µL of Genomic Lysis buffer in a fume hood to the filtrate in the collection tube from step 4. Mix well.
Note
Genomic Lysis buffer should have beta-mercaptoethanol added to a final dilution of 0.5% (v/v) ie Amount500 µL per Amount100 mL buffer. Beta-mercaptoethanol is a strong reducing agent added to clean tannins and other polyphenolics found in plant extracts. Please refer to material SDS and use appropraite PPE.



DNA purification
DNA purification
9m 30s
9m 30s
Transfer Amount800 µL of the mixture from step 10 to a Zymo-Spin IICR column in a collection tube and centrifuge at Centrifigation10000 x g, 18°C, 00:01:00 . Discard flow through from the collection tube and repeat with remaining mixture.

1m
Add Amount200 µL DNA Pre-Wash buffer to the Zymo-Spin IICR Column in a new collection tube and centrifuge Centrifigation10000 x g, 18°C, 00:01:00 .

1m
Add Amount500 µL g-DNA Wash Buffer to the Zymo-Spin IICR Column and centrifuge Centrifigation10000 x g, 18°C, 00:01:00 .

1m
Transfer the Zymo-Spin IICR column to a clean Amount1.5 mL centrifuge tube and add Amount100 µL * DNA Elution buffer directly to the column matrix. Centrifuge Centrifigation10000 x g, 18°C, 00:00:30 to elute the DNA.

Note
* If samples exhibit a low biomass it's recommended that this volume be reduced to Amount50 µL in order to concentrate DNA recovered.


30s
Place a Zymo-Spin III-HRC Filter in a clean collection tube and add Amount600 µL prep solution. Centrifuge Centrifigation8000 x g, 18°C, 00:03:00 . Discard flow through and place in clean labelled 1.5 ml microcentrifuge tube.

3m
Transfer Eluted DNA from step 14 to a prepared Zymo-Spin III-HRC Filter and centrifuge at exactly Centrifigation16000 x g, 18°C, 00:03:00 .

3m
The filtered DNA is now suitable for PCR and other downstream applications.