TITLE: The extraction of cross-kingdom biofilm DNA using Zymo Reasearch Quick-DNA Fecal/Soil Microbe Kits.
AUTHORS: Lindsay Kate Newbold, Daniel S Read, Joetay, Jonathan Warren, kerry.walsh

[DESCRIPTION]
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. 

[BEFORE_START]
Add 500 µL beta-mercaptoethanol per 100ml Genomic Lysis Buffer.


[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.

[STEPS]
SECTION: Sample Preparation
1. In field samples are taken by the Environment Agency, and 5 mL  biofilm suspension preserved in equal amounts of preservation buffer: 3.5 Molarity (M) ammonium sulphate, 17 millimolar (mM) sodium citrate, 13 millimolar (mM) EDTA. Samples are frozen in 
 
 prior to laboratory preparation.

SECTION: Sample Preparation
2. Once back in the laboratory field preserved samples are first thawed and then vortexed to create a homogenous mixture.
SECTION: Sample Preparation
3. Samples are pelleted at 3000 x g, 5 °C  for 15 min 

SECTION: Sample Preparation
4. Ensure samples are fully pelleted, then remove 500 µL of buffer to separate microcentrifuge tube.
 
                                                                                          
Remaining buffer is gently aspirated to avoid disrupting pellet.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

SECTION: Sample Preparation
5. Pellet is resuspended in  500 µL of reserved preservation buffer, and stored in the same fully labelled microcentrifuge tube. Preserved sample is then frozen at -80 °C  prior to analysis.

SECTION: Sample Lysis
6. Thaw sample to room temperature, vortex to resuspend pellet and add 100 µL  to ZR BashingBead Lysis tube. Add 900 µL  DNA/RNA shield, and lyse for 40 min  on a horizontal vortex mixer (Vortex Genie).

SECTION: Sample Lysis
7. Briefly spin for 10000 x g, 1 min, 18 °C . Add 40 µL proteinase K, resuspend and mix through pipetting  and incubate at 65 °C for 20 min .

SECTION: Sample Lysis
8. Centrifuge ZR BashingBead tube at 10000 x g, 5 min, 18 °C to pellet lysate.

SECTION: Sample Lysis
9. Transfer up to 400 µL  supernatant to a Zymo-Spin III-F Filter in a collection tube and centrifuge at 8000 x g, 1 min, 18 °C .

SECTION: DNA purification
11. Transfer 800 µL  of the mixture from step 10 to a Zymo-Spin IICR column in a collection tube and centrifuge at 10000 x g, 1 min, 18 °C . Discard flow through from the collection tube and repeat with remaining mixture.

SECTION: Sample Lysis
10. Add 1200 µL  of Genomic Lysis buffer in a fume hood to the filtrate in the collection tube from step 4. Mix well.
 


SECTION: DNA purification
12. Add 200 µL  DNA Pre-Wash buffer to the Zymo-Spin IICR Column in a new collection tube and centrifuge 10000 x g, 1 min, 18 °C .

SECTION: DNA purification
13. Add 500 µL  g-DNA Wash Buffer to the Zymo-Spin IICR Column and centrifuge 10000 x g, 1 min, 18 °C  .

SECTION: DNA purification
14. Transfer the Zymo-Spin IICR column to a clean 1.5 mL  centrifuge tube and add 100 µL * DNA Elution buffer directly to the column matrix. Centrifuge 10000 x g, 30 s, 18 °C  to elute the DNA.

 

SECTION: DNA purification
15. Place a Zymo-Spin III-HRC Filter in a clean collection tube and add 600 µL  prep solution. Centrifuge 8000 x g, 3 min, 18 °C . Discard flow through and place in clean labelled 1.5 ml microcentrifuge tube.

SECTION: DNA purification
16. Transfer Eluted DNA from step 14 to a prepared Zymo-Spin III-HRC Filter and centrifuge at exactly 16000 x g, 3 min, 18 °C.

SECTION: DNA purification
17. The filtered DNA is now suitable for PCR and other downstream applications.