Zymo Research ZymoBIOMICS D4300T User manual

For Research Use Only Ver. 1.4.1
90
ZymoBIOMICS™ DNA Miniprep Kit
Catalog Nos. D4300T, D4300, & D4304
Highlights
• Validated Unbiased for Microbiome Measurements: Unbiased cellular lysis validated using the
ZymoBIOMICS Microbial Community Standard.
• Inhibitor-Free DNA from Any Sample: Isolate ultra-pure DNA ready for any downstream application.
• Certified Low Bioburden: Boost your detection limit for low abundance microbes.
• Simple Workflow: Simply bead-beat sample, purify via spin-column, and filter to remove PCR inhibitors.
No precipitation or lengthy incubations!
Contents
Product Contents & Specifications ................................. 1
Product Description ..................................................... 2-3
Protocol ........................................................................ 4-5
Appendices
A. Sample Collection ............................................... 6
B. Application Notes ........................................... 7-10
C. Standardize Sample Preparation with
ZymoBIOMICS™ Microbial Standards ....... 11-12
D. Troubleshooting ........................................... 12-13
Ordering Information ..................................................... 14
INSTRUCTION MANUAL

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Product Contents
ZymoBIOMICS™ DNA Miniprep Kit
(Kit Size)
D4300T
(5 preps.)
D4300
(50 Preps.)
D4304
(50 Preps.)
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm)
5
50
-
ZymoBIOMICS™ Lysis Solution
4 ml
40 ml
-
ZymoBIOMICS™ DNA Binding Buffer1
6 ml
100 ml
100 ml
ZymoBIOMICS™ DNA Wash Buffer 1
2 ml
50 ml
50 ml
ZymoBIOMICS™ DNA Wash Buffer 2
5 ml
60 ml
60 ml
ZymoBIOMICS™ DNase/RNase Free Water
1 ml
10 ml
10 ml
ZymoBIOMICS™ HRC Prep Solution
3 ml
30 ml
30 ml
Zymo-Spin™ III-F Filters
5
50
50
Zymo-Spin™ III-HRC Filters
5
50
50
Zymo-Spin™ IICR Columns
5
50
50
Collection Tubes
20
200
200
Instruction Manual
1
1
1
Storage Temperature - Store all kit components (i.e., buffers, columns) at room temperature.
Note - Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely tested
on a lot-to-lot basis to ensure they provide maximal performance and reliability.
Specifications
• Sample Sources – Bacterial (including endospores)1, fungal, protozoan, algal, viral, mitochondrial, and
host DNA is efficiently isolated from ≤ 200 mg of mammalian feces, ≤ 250 mg soil, and 50 – 100 mg (wet
weight) of bacterial/fungal cells2, biofilms, and water3.
• Bead Beating System – The innovative ZymoBIOMICS™ lysis system enables complete
homogenization/disruption of the microbial cells walls and accurate microbial DNA analysis, free of bias.
To ensure unbiased lysis, calibration of each bead-beating device is recommended by using the
ZymoBIOMICS™ Microbial Community Standard (see Appendix C).
• DNA Purity – High quality, inhibitor-free DNA is eluted with ZymoBIOMICS™ DNase/RNase Free Water
and is suitable for all downstream applications including PCR and Next-Generation Sequencing.
• DNA Integrity – On average, post bead beating, genomic DNA is between 15-20 kb depending on the
initial quality of the sample, making it amenable to Next-Generation Sequencing platforms requiring high
molecular weight DNA. For optimal DNA integrity, collect samples in DNA/RNA Shield™4.
• DNA Recovery – Up to 25 µg total DNA can be eluted into 100 µl (50 µl minimum).
• Bioburden – A single preparation is guaranteed to contain less than 3 bacterial genomic copies per µl of
eluate as determined by quantitative amplification of the 16S rRNA gene when eluted using 100 µl water.
Individual components (sold separately) are not certified low-bioburden.
• Equipment – Microcentrifuge, vortex/Disruptor Genie®, high speed cell disrupter (recommended).
Satisfaction of all Zymo
Research products is
guaranteed. If you should
be dissatisfied with this
product please call
1-888-882-9682.
1 See endospore lysis
efficiency data in Appendix
B.
2 This equates to
approximately 109 bacterial
cells and 108 yeast cells.
3 For water samples, filter
using desired filter (not
provided). Cut the filter into
small pieces and place into
ZR BashingBead™ Lysis
Tube (0.1 & 0.5 mm).
Alternatively, up to 250 µl
water can be processed
directly.
4 DNA/RNA Shield™
provides an accurate
molecular signature of the
sample at the time of
collection by preserving
nucleic acids at ambient
temperature and inactivating
organisms including
infectious agents (see
Appendix A).

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Product Description
The ZymoBIOMICS™ DNA Miniprep Kit is designed for purifying DNA from a wide array of sample
inputs (e.g. feces, soil, water, biofilms, etc.), that is immediately ready for microbiome or metagenome
analyses. The ZymoBIOMICS™ innovative lysis system eliminates bias associated with unequal lysis
efficiencies1 of different organisms (e.g. Gram-negative/positive bacteria including endospores2, fungi,
protozoans, algae, etc.), making it ideal for microbial community profiling. Unbiased mechanical lysis of tough
microbes is achieved by bead beating with the innovative ultra-high density BashingBeads™ and validated
using the ZymoBIOMICS™ Microbial Community Standard3, as shown in Figure 3. In addition, the
ZymoBIOMICS™ DNA Miniprep Kit is equipped with Zymo Research’s proprietary OneStep™ PCR Inhibitor
Removal technology enabling PCR from the most PCR prohibitive environmental samples rich in humic and
fulvic acids, tannins, melanin, and other polyphenolic compounds. Coupling state-of-the-art lysis technology
with Zymo-Spin™ Technology results in superior yields of ultra-pure DNA ideal for all downstream applications
including PCR, arrays, 16S rRNA gene sequencing, and shotgun sequencing4.
Innovation. Pure & Simple.™
Superior Yields
Zymo- Supplier
L BIOMICS
™
Q1 P Q2
Bias free lysis
Using ZymoBIOMICS™
Innovative Lysis System.1
Filter debris from lysate
using Zymo-Spin™ IV.
Superior yields with Zymo
Spin™ IIIC-Z. Simply Bind,
Wash, and Elute.
Complete PCR inhibitor
removal with Zymo Spin™
IV-HRC-Z
Superior yields.
Simply Bind, Wash, and
Elute from the Zymo-Spin™
IIIC-Z.
Complete PCR Inhibitor
Removal by simply filtering
the eluate with a Zymo-
Spin™ HRC.
1Chemical, enzymatic, and
inferior lysis matrices
(beads) lead to unrealistic
representation of organisms
in downstream metagenomic
analyses that is not
reflective of actual
abundance.
2See endospore lysis
efficiency data in Appendix
B.
3 For more information on
the ZymoBIOMICS™
Microbial Community
Standard (D6300) &
ZymoBIOMICS™ Microbial
Community DNA Standard
(D6305), see Appendix C.
4 DNA is predominately
15-20 kb and amenable to
Next-Generation
Sequencing techniques
requiring high molecular
weight DNA.
Zymo Research offers a full
suite of ZymoBIOMICS™
Services for reliable,
accurate microbial and
metagenomic analyses.
Services include: Microbial
Composition Profiling, Novel
Microbe Identification, and
Customizable
Bioinformatics.
For details visit us at:
http://www.zymoresearch.com/
services/metagenomics
Or contact us at:
[email protected]
Ultra-pure DNA from Inhibitor Rich Samples
Figure 1. The ZymoBIOMICS™ DNA Miniprep Kit provides inhibitor-free DNA even when
challenged with extremely inhibitor rich samples. Real-time PCR was used to evaluate eluates
recovered using the ZymoBIOMICS™ DNA Miniprep Kit, and kits from Suppliers Q1, P, and
Q2. Reaction volumes consisted of either 10% or 35% of the eluate from each kit to detect the
presence of PCR inhibitors. Each reaction contained 25 ng of Brettanomyces DNA. Delayed
and/or no amplification indicates PCR inhibition from inefficient inhibitor removal.
Figure 2. The ZymoBIOMICS™ DNA Miniprep Kit provides superior yields when compared to
Suppliers Q1, P, and Q2. 80 mg of feces was processed using each kit according to the
manufacturers’ recommended protocol. DNA was eluted using 100 µl ZymoBIOMICS™
DNase/RNase Free Water. 6 µl of each sample was visualized in a 1.0% (w/v)
agarose/ethidium bromide gel. Samples were processed in triplicate. L is a 1Kb ladder.

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Theoretical ZymoBIOMICS™ HMP Protocol Supplier Q1 Supplier Q2
DNA Miniprep Kit
Figure 3. A) The ZymoBIOMICS™ DNA Miniprep Kit provides unbiased representation of the organisms extracted from the
ZymoBIOMICS™ Microbial Community Standard. DNA was extracted from ZymoBIOMICS™ Microbial Community Standard using
four different DNA extraction methods (ZymoBIOMICS™ DNA Miniprep Kit, Human Microbiome Project Protocol, Supplier Q1, and
Supplier Q2) and analyzed using 16S rRNA gene sequencing. 16S rRNA genes were amplified with primers targeting v3-4 region and
the amplicons were sequenced on Illumina® MiSeq™ (2x250bp). Overlapping paired-end reads were assembled into complete
amplicon sequences. The composition profile was determined based on sequence counts after mapping amplicon sequences to the
known 16S rRNA genes of the eight different bacterial species.
B) The ZymoBIOMICS™ DNA Miniprep Kit reliably isolates DNA from even the toughest to lyse Gram positive organisms,
enabling unbiased analyses of microbial community compositions. There is a significant increase in yield and Gram-positive
bacterial abundance when DNA was isolated using the ZymoBIOMICS™ DNA Miniprep Kit. Correlated with the results in Figure 3A, it
can be concluded that unbiased DNA isolation was achieved. DNA was extracted from 200 µl of human feces suspended in PBS
(10 % m/v) using four different DNA extraction methods (ZymoBIOMICS™ DNA Miniprep Kit, Human Microbiome Project Protocol,
Supplier Q1, and Supplier Q2) and analyzed using 16S rRNA gene sequencing. 16S rRNA genes were amplified with primers targeting
v3-4 region and the amplicons were sequenced on Illumina® MiSeq™ (2x250bp). Overlapping paired-end reads were assembled into
complete amplicon sequences. Amplicon sequences were profiled with Qiime using Greengenes 16S rRNA gene database
(gg_13_8).
DNA is ready for all downstream
applications.
A) Bias Free Microbial DNA Extraction Using ZymoBIOMICS™ DNA Miniprep Kit
Validated with the ZymoBIOMICS™ Microbial Community Standard
B) Bias Free Microbial DNA Extraction Using ZymoBIOMICS™ DNA Miniprep Kit
From Human Stool
ZymoBIOMICS™ HMP Protocol Supplier Q1 Supplier Q2
DNA Miniprep Kit

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Protocol
1. Add sample to a ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm). Add
750 µl ZymoBIOMICS™ Lysis Solution to the tube and cap tightly.
Note: For samples stored and lysed in DNA/RNA Shield™ Lysis Tubes, do not add
ZymoBIOMICS™ Lysis Solution and proceed to Step 2.
Sample Type
Maximum Input
Feces
200 mg
Soil
250 mg
Liquid Samples1 and Swab Collections2
250 µl
Cells (isotonic buffer, e.g. PBS)
50-100 mg (wet weight)
(109 bacterial and 108 yeast cells)
Samples in DNA/RNA Shield™,3
≤ 1 ml
2. Secure in a bead beater fitted with a 2 ml tube holder assembly and
process at maximum speed for ≥ 5 minutes.
Note: Processing time will vary based on sample input and bead beater. Times
may be as little as 5 minutes when using high-speed cell disrupters (FastPrep®
-24) or as long as 20 minutes when using lower speeds (e.g., Disruptor Genieâ).4
3. Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a
microcentrifuge at ≥ 10,000 x g for 1 minute.
4. Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter in a
Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the
Zymo-Spin™ III-F Filter.
5. Binding preparation:
Feces and All Non-Soil Samples
OR
Soil Samples
Add 1,200 μl of ZymoBIOMICS™ DNA
Binding Buffer to the filtrate in the
Collection Tube from Step 4. Mix well.
Add 800 μl of ZymoBIOMICS™ DNA
Binding Buffer and 400 μl of 95%
ethanol to the filtrate in the Collection
Tube from Step 4. Mix well.
6. Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IICR Column
in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
7. Discard the flow through from the Collection Tube and repeat Step 6.
8. Add 400 µl ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™
IICR Column in a new Collection Tube and centrifuge at 10,000 x g for
1 minute. Discard the flow-through.
9. Add 700 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™
IICR Column in a Collection Tube and centrifuge at 10,000 x g for
1 minute. Discard the flow-through.
For Technical Assistance:
1-888-882-9682 or E-mail
[e-mail]
1For water samples, filter
using desired filter (not
provided). Cut the filter into
small pieces and place into
ZR BashingBead™ Lysis
Tubes (0.1 & 0.5 mm).
2Swabs can also be cut or
broken, then placed directly
in bead beating tube. For
more information on
processing swab samples,
see Appendix B.
3 Up to 1 ml of sample in
DNA/RNA Shield can be
processed directly in ZR
BashingBead™ Lysis Tube.
Adjust final volume to 1 ml
with ZymoBIOMICS™ Lysis
Solution or DNA/RNA
Shield, if necessary.
4For optimal lysis efficiency
and unbiased profiling, all
bead beater devices beyond
those validated by Zymo
Research should be
calibrated using the
ZymoBIOMICS™ Microbial
Community Standard (see
Appendix C).

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10. Add 200 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR
Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
11. Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube
and add 100 µl (50 µl minimum) ZymoBIOMICS™ DNase/RNase Free Water
directly to the column matrix and incubate for 1 minute. Centrifuge at
10,000 x g for 1 minute to elute the DNA5, 6.
12. Place a Zymo-Spin™ III-HRC Filter in a new Collection Tube and add 600 µl
ZymoBIOMICS™ HRC Prep Solution. Centrifuge at 8,000 x g for 3 minutes.
13. Transfer the eluted DNA (Step 11) to a prepared Zymo-Spin™ III-HRC Filter in
a clean 1.5 ml microcentrifuge tube and centrifuge at exactly 16,000 x g for
3 minutes.
The filtered DNA is now suitable for PCR and other downstream applications.
For Technical Assistance:
1-888-882-9682 or E-mail
[e-mail]
5 In some cases a brown-
colored pellet may form at
the bottom of the tube after
centrifugation. Avoid this
pellet when collecting the
eluted DNA.
6 If fungi or bacterial cultures
were processed; the DNA is
now suitable for all
downstream applications.

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Appendix A
Sample Collection
For high quality reproducible microbiomics data, DNA/RNA Shield™ is recommended for sample collection to avoid bias or erroneous
results due to compositional changes from nucleic acid degradation or microbial growth. DNA/RNA Shield™ provides an unbiased
molecular snapshot of the sample at the time of collection by preserving nucleic acids at ambient temperature and inactivating organisms
including infectious agents. Samples can be stored and transported easily and safely with DNA/RNA Shield™ and is ideal for applications
such as PCR, 16S rRNA gene sequencing, and shotgun metagenomic sequencing. DNA/RNA Shield™ can preserve nucleic acids in nearly
any sample including feces, soil, saliva, blood, and tissues.
DNA/RNA Shield™ - Lysis Tube (Microbe) – Simply add sample, seal and store at ambient temperature. The Lysis Tube is immediately
ready for bead beating, thereby streamlining the collection to extraction transition. (Cat. No. R1103)
DNA/RNA Shield™ – Fecal Collection Tube – The collection device is specifically designed for easy collection and stabilization of feces.
Includes a scoop built for collecting 1 gram of feces (or any other sample such as saliva or soil). (Cat. No. R1101)
DNA/RNA Shield™ – Swab Collection Tube – Easy collection of biological samples; swab has breakable tip to allow for easy sample
collection and removes the need to dispose of a potentially biohazardous swab material. (Cat. No. R1106 & R1107)
Figure 4. A) Nucleic acids in stool are effectively stabilized in DNA/RNA Shield™ at room temperature. Graph shows spike-in DNA and RNA controls from
stool purified at the indicated time points and analyzed by (RT)qPCR. Controls: HSV-1 and HIV (AcroMetrix™, Life Technologies).
B) Microbial composition of stool is unchanged after one month at ambient temperature with DNA/RNA Shield™. Stool samples suspended in DNA/RNA
Shield™ and stored at room temperature were compared to stool without preservative for one month. They were sampled at the indicated time points and
processed with ZymoBIOMICS™ DNA Miniprep Kit. The extracted DNA was then subjected to microbial composition profiling via 16S rRNA gene targeted
sequencing. Graphs show both phylum composition (left) and genus composition (right). Samples stored with DNA/RNA Shield™ had a constant microbial
composition while the samples stored without shifted dramatically.
C) Viruses, bacteria and yeast are effectively inactivated by DNA/RNA Shield™. Samples containing the infectious agent (viruses, bacteria, yeast) were
treated with DNA/RNA Shield™ or mock (PBS) treated for 5 minutes. Titer (PFU) was subsequently determined by plaque assay. Validated by: Influenza A - D.
Poole and Prof. A. Mehle, Department of Medical Microbiology and Immunology, University of Wisconsin, Madison; Ebola (Kikwit) - L. Avena and Dr. A.
Griffiths, Department of Virology and Immunology, Texas Biomedical Research Institute; HSV-1/2.
A) DNA/RNA Shield™ Preserves
Nucleic Acids at Room Temperature
C) DNA/RNA Shield™ Inactivates Pathogens
for Safe Transport and Storage
B) DNA/RNA Shield™ Preserves Microbial Composition at Room Temperature

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Appendix B
Application Notes
DNA/RNA Shield™ Lysis Tubes (Microbe) (Cat. No. R1103)
1. Collect sample directly into the DNA/RNA Shield™ Lysis Tube (Microbe).
2. Directly proceed to Step 2 of the protocol (page 4) and bead beat in the DNA/RNA Shield™ Lysis Tube
(Microbe) according to provided recommendations.
3. Proceed with the remaining protocol as written.
DNA Viruses
For unbiased metagenomics analysis of viruses, incorporating a Proteinase K digestion prior to bead beating is
recommended.
1. Following Step 2 (page 4) add 5% (v/v) of Proteinase K (Cat. No. D3001-2-5) to the lysate within the
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) and incubate for 30 minutes at 55˚C.
2. Proceed to Step 3 (page 4) and continue with the remaining protocol as written.
Cheese and Protein Rich Biofluids (e.g. Milk, Sputum, Saliva, Spinal Fluid, Blood, and Serum)
1. Add ≤ 0.4 g of cheese or ≤ 200 µl of biofluid to the ZR BashingBead
™
Lysis Tubes (0.1 & 0.5 mm).
Add 750 µl of ZymoBIOMICS
™
Lysis Solution.
2. Add 20 µl of Proteinase K (20 µg/µl) (cat. no. D3001-2-5) to the ZymoBIOMICS™ Lysis Tubes (0.1 & 0.5 mm)
and incubate for 30 minutes at 55˚C.
3. Continue to Step 2 (page 4) and proceed with the protocol as written.
Plant Tissue (Leaves and other plant material)
Plant tissues such as leaves and roots contain DNA sources within the host tissue that can overwhelm 16S rRNA
gene targeted sequencing (from both mitochondria & chloroplast). Microbes must be removed from the plant
surface to exclude host tissue from the bead beating process.
(A) Plant tissue – Centrifugation of cells
1. Suspend plant tissue in isotonic solution (e.g. PBS) and gently sonicate or vortex briefly.
2. Remove plant tissue from solution and centrifuge at 15,000 x g for 10 minutes to pellet the cells.
3. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 300 µl of pellet.
4. Add ZymoBIOMICS™ Lysis Solution to the cells to a final volume of 1 ml and mix to resuspend. Transfer the
mixture to the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) and proceed to Step 2 (page 4).
(B) Plant tissue – Filtration of cells
1. Place plant tissue in a submerging volume of PBS inside of a conical tube and gently sonicate or vortex briefly.
Remove plant tissue from liquid volume.
2. Filter liquid using a 0.22 µm filter (not provided).
3. Cut the filter and place directly into the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) and proceed to
Step 1 (page 4).

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(C) Plant root – Lysis of surface microbes
1. Cut root into small pieces and place directly into ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) with 750 µl of
ZymoBIOMICS™ Lysis Buffer.
2. Lysis should be performed with a lower speed bead beating device (e.g. vortex adapter for 20 minutes) to avoid the
host tissue contamination.
3. Continue to Step 3 (page 4) and proceed with the remaining protocol as written.
Water/Air Samples
1. Filter samples using desired filter (not provided) prior to Step 1 (page 4).
2. Cut the filter into small pieces and place them inside the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm),
and add 750 µl of ZymoBIOMICS Lysis Solution.
3. Continue to Step 2 (page 4) and proceed with the remaining protocol as written.
Lytic Enzymes
Lytic enzymes, such as Lysozyme, Lysostaphin, MetaPolyzyme, etc. can be used with this kit using the following:
(A) Enzymatic lysis followed by bead beating:
1. Perform enzymatic digestion under manufacturer’s recommended conditions (temperature/time/concentration).
Note: If sample is stored in DNA/RNA Shield, perform the following:
a. Centrifuge sample at ≥ 10,000 x g for 1 minute.
b. Transfer supernatant to a ZR BashingBead Lysis Tube (0.1 & 0.5 mm), to be used in Step 2, below.
c. Re-suspend pellet in a buffer suitable for enzymatic treatment (ex. PBS or other isotonic solution).
2. Transfer the digestion mixture to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm).
3. Add 750 µl ZymoBIOMICS™ Lysis Solution.
Note: For samples in DNA/RNA Shield, raise to a final volume of 1 ml with DNA/RNA Shield.
4. Proceed to Step 2 (page 4) and continue with the remaining protocol as written.
(B) Enzymatic lysis only (no bead beating):
1. Perform enzymatic digestion under manufacturer’s recommended conditions (temperature/time/concentration).
Note: If sample is stored in DNA/RNA Shield, perform the following:
a. Centrifuge sample at ≥ 10,000 x g for 1 minute.
b. Transfer supernatant to a clean microcentrifuge tube, to be used in Step 2.
c. Re-suspend pellet in a buffer suitable for enzymatic treatment (ex. PBS or other isotonic solution).
2. Raise the volume of sample to 400 µl with ZymoBIOMICS™ Lysis Solution.
3. Continue to Step 4 (page 4) and proceed with the remaining proceed as written.
Hair, Feather, and Nail Samples:
1. To ≤ 25 mg sample, add 90 µl Water, 90 µl Solid Tissue Buffer (Blue) (Cat. No. D4068-2-6), 10 µl 1M DTT,
and 10 µl Proteinase K (Cat. No. D3001-2-5) in a microcentrifuge tube.
2. Mix thoroughly or vortex 10-15 seconds and then incubate the tube at 55ºC overnight.
3. Transfer lysate to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and then add 750 µl ZymoBIOMICS™
Lysis Solution.
4. Continue to Step 2 (page 4) and proceed with the remaining protocol as written.

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Tissue and Insect Samples
Tissue and Insect samples can be processed three different ways, depending on the sample type and the
equipment available. The recommendations are listed next to the options below:
(A) Proteinase K - Tissue
1. Add up to 15 mg of tissue to a 1.5 ml microcentrifuge tube, then add a solution of 95 µl water, 95 µl Solid Tissue
Buffer (Blue) (Cat. No. D4068-2-6) and 10 µl Proteinase K (Cat. No. D3001-2-5). Incubate for at least 1 hour at
55° C or until tissue clarifies (samples can be incubated overnight without affecting DNA quality).
2. Transfer digestion to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and add 750 µl of ZymoBIOMICS™ Lysis
Solution.
3. Proceed to Step 2 (page 4) and continue with the protocol as written.
(B) Bead beating -Tissue and Insect
1. Add up to 15 mg of tissue/insect sample in a ZR BashingBead™ Lysis Tube (2.0 mm) (Cat. No. S6003-50) with
750 µl of ZymoBIOMICS™ Lysis Solution.
2. Secure in a bead beater fitted with a 2 ml tube holder assembly and process at maximum speed for ≥ 5 minutes.
Note: Processing time will vary based on sample input and bead beater. Times may be as little as 5 minutes when using
high-speed cell disrupters (FastPrep® -24) or as long as 20 minutes when using lower speeds (e.g., Disruptor Genie®).
3. Transfer the entire lysate to the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm), proceed to Step 2 (page 4), and
continue with protocol as written.
(C) Mortar & Pestle - Tissue and Insect
1. Homogenize up to 15 mg tissue/insect sample with a mortar and pestle while submersed in liquid nitrogen.
2. Transfer the entire sample into the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and add 750 µl of
ZymoBIOMICS™ Lysis Solution.
3. Proceed to Step 2 (page 4) and continue with the protocol as written.
Samples Collected with Swabs
(A) Directly process swab
1. Directly break swab at breakpoint or cut the swab into a ZR BashingBead Lysis Tube (0.1 & 0.5 mm).
2. Proceed to Step 1 (page 4) and continue with the protocol as written.
(B) Indirectly process swab
1. Vortex the swab in the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) with 750 µl of ZymoBIOMICS™ Lysis
Solution for 30 seconds to transfer the microbes into solution.
2. Remove the swab and proceed to bead beating in Step 2 (page 4).
Figure 5. Phylum composition of a simulated microbial community
when bead beating was performed with and without the presence of
a Puritan HydraFlock® sterile flocked collection device placed in a ZR
BashingBead Lysis Tube and processed at maximum speed
(6.5 m/s) for 5 minutes. The extracted DNA was then subjected to
microbial composition profiling via 16S rRNA gene targeted
sequencing. Experiment was performed in technical duplicates

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Bacterial Endospore Lysis
ZymoBIOMICS DNA Miniprep Kit is capable of effectively lysing bacterial endospores, and also achieves higher
yield when compared to competition.
Urine
(A) Pelleting cells from fresh/frozen urine
1. Pellet the bacterial cells by centrifuging the urine at 15,000 x g for 10 minutes.
2. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 400 µl of pellet.
3. Add ZymoBIOMICS™ Lysis Solution to a final volume of 800 µl and then transfer the mixture to a
ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Proceed to Step 2 (page 4) and continue with the protocol as written.
(B) Pelleting cells from stabilized urine
1. Add 70 µl Urine Conditioning Buffer (Cat. No. D3061-1-140) for every 1 ml of urine and mix well by vortexing.
Note: Urine stabilized by the Urine Conditioning Buffer can be stored for up to 1 month at ambient temperature. When samples
are ready to be processed, mix well by vortexing, and proceed to Step 2.
2. Centrifuge at 3,000 x g for 15 minutes.
3. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 400 µl of pellet.
4. Add ZymoBIOMICS™ Lysis Solution to a final volume of 800 µl and then transfer the mixture to a
ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Proceed to Step 2 (page 4) and continue with the protocol as
written.
Figure 6. DNA Extractions were performed using the ZymoBIOMICS® DNA Kit and DNeasy
PowerSoil with 6 x 108 B. subtilis CFU. DNeasy PowerSoil reovered 0.62 ng/µl DNA, while the
ZymoBIOMICS® DNA Kit was capable of recovering 9.67 ng/µl in a 50 µl elution volume.
Extractions were performed in triplicate and quantified via Qubit.
Figure 7. Phylum composition of urine preserved in Urine
Conditioning buffer™ (UCB™), which preserves the microbial
composition of urine with simulated stool contamination for a
month at room temperature. Urine with UCB™ added (Zymo
Research, D3061-1-160) was stored at room temperature and
analyzed over a month period. At the indicated time points (0
Days, 2 weeks, and 1 month), DNA was extracted using the
ZymoBIOMICS™ DNA Miniprep Kit. The extracted DNA was
then subjected to microbial composition profiling via 16S rRNA
gene targeted sequencing. Experiment was performed in
technical duplicates.
0 Day
2 Weeks
1 Month

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Appendix C
Standardize Sample Preparation with ZymoBIOMICS™ Microbial Community Standards
The ZymoBIOMICS™ Microbial Community Standard (Cat. No. D6300) is a mock microbial community of defined and well characterized
composition making it the perfect control for all microbiome profiling and metagenomics analyses.
It is ideal for assessing bias of DNA extraction methods since it contains three easy-to-lyse Gram-negative bacteria (e.g. Escherichia coli),
five tough-to-lyse Gram-positive bacteria (e.g. Listeria monocytogenes), and two tough-to-lyse yeasts (e.g. Saccharomyces cerevisiae).
Bead Beating Device Calibration Protocol:
Zymo Research suggests calibrating bead beating devices with the ZymoBIOMICS™ Microbial Community Standard in order to ensure bias
free microbial extraction. For Disruptor Genieâ, vortex adapters, and vortex lysis we suggest a time course ranging from 10-45 minutes with
the vortex at maximum speed. For high speed cell disruptors such as the MP FastPrep -24â we suggest a time course at maximum speed
with a range of 3-10 minutes. The resulting DNA should be evaluated by quantifying DNA yield and changes in microbial profile at each
time point. The bead beating time that yields a profile that closely matches the theoretical composition should become standard operating
procedure for the bead beating device.
ZymoBIOMICS™ Microbial Community DNA Standard (Cat. No. D6305) is a mixture of genomic DNA extracted from pure cultures of
eight bacterial and two fungal strains. Genomic DNA from each culture was quantified before mixing. The ZymoBIOMICS™ Microbial
Community Standard allows for assessment of bias from library preparation, sequencing, and bioinformatics analysis.
It serves perfectly as a microbial standard for benchmarking the performance of microbiomics or metagenomics analyses, including those
provided by a 3rd party.
Species
GC
%
Gram
Stain
gDNA
Abun.
(%)
Pseudomonas
aeruginosa
66.2
-
12
Escherichia coli
56.8
-
12
Salmonella enterica
52.2
-
12
Lactobacillus fermentum
52.8
+
12
Enterococcus faecalis
37.5
+
12
Staphylococcus aureus
32.7
+
12
Listeria monocytogenes
38.0
+
12
Bacillus subtilis
43.8
+
12
Saccharomyces
cerevisiae
38.4
Yeast
2
Cryptococcus
neoformans
48.2
Yeast
2
gDNA by Shotgun Sequencing
16S Counts by 16S Sequencing
Accurate composition for reliable use to evaluate shotgun seq. and 16S rRNA seq.
Figure 8. Characterization of the microbial composition of the two ZymoBIOMICS™ standards with shotgun metagenomic sequencing (left panel) and 16S rRNA gene
targeted sequencing (right panel). The measured composition of the two standards agrees with the theoretical/designed composition. “DNA Standard” represents
ZymoBIOMICS™ Microbial Community DNA Standard (DNA version) and “Microbial Standard” represents ZymoBIOMICS™ Microbial Community Standard (cellular
version). Genomic DNA composition by shotgun sequencing was calculated based on counting the amounts of raw reads mapped to each genome. 16S composition
by 16S rRNA gene targeted sequencing was calculated based on counting the amount of 16S raw reads mapped to each genomes.

Page 12
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Appendix D
Troubleshooting:
For Technical Assistance, please contact 1-888-882-9682 or E-mail [e-mail].
Problem Possible Causes and Suggested Solutions
• Clean workspace, centrifuge, and pipettes with 10% bleach to
routinely to avoid contamination.
• Use of kit in exposed environment without proper filtration.
Check pipettes, pipette tips, microcentrifuge tubes, workspace,
etc. for contamination.
Background Contamination
• Make sure bags of columns and buffer bottles are properly
sealed for storage. Use of these outside a clean room or hood
can result in contamination.
A) Use ZymoBIOMICS™ Microbial Standards for assessing GC-Bias
in Shotgun Metagenomics
B) Perfect for tracking PCR Chimera in 16S
rRNA Gene Sequencing
Figure 9.
A) Library preparation for shotgun metagenomic sequencing was performed in two different ways: one by supplier I and one by an in-house method. Shotgun
sequencing was performed on Illumina® MiSeq™ with paired-end sequencing (2 x 150 bp). Raw reads were mapped to the 10 microbial genomes to evaluate the
potential effect of GC content on sequencing coverage. Normalized coverage was calculated by normalization by the average sequencing coverage of each
genome
B) PCR chimera increases with PCR cycle number in the library preparation process of 16S rRNA gene targeted sequencing. 20 ng ZymoBIOMICS™ Microbial
Community Standard was used a template. The PCR reaction was performed with ZymoBIOMICS™ PCR Premix and with primers that target v3-4 region of 16S
rRNA gene. Chimera rate in percentage was determined with Uchime and using the 16S rRNA gene of the 8 bacterial strains in the standard as reference PCR.
chimera sequences were defined as sequences resulted from the recombination/hybridization of different template sequences.

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Lysis Methods
• When using a Disruptor Genieâ, vortex adapter, vortex, or
similar processing times will vary. Suggested beat beating time
is anywhere from 5-20 minutes. Calibrate bead beating times
to your particular device and application by testing several
different time points before using precious samples.
(Suggested times to test: 10, 20, and 30 minutes.) See
Appendix C for details.
• When using high-speed bead bashing devices (e.g. Bertin
Percellys, MP – FastPrep24) run max speed for 5 minutes to
ensure unbiased lysis.
Incomplete Debris Removal
• For high density samples, ensure lysate is centrifuged properly
to pellet insoluble debris following bead beating. Ensure that
none of the debris is transferred to the Zymo-Spin™ III-F
Filter in the next step.
Low DNA Yield
Input
• If the lysate does not pass through the column or is extremely
viscous, use less input material. Too much sample input can
cause cellular debris to overload the column and insufficient
flow
• Consult the Sample Input Table on Page 4 for information on
your particular input limit based on sample.
Binding Step
• Ensure that the ZymoBIOMICS™ DNA Binding Buffer is
completely mixed with lysate before loading onto the column.
Improperly mixed samples can lead to poor DNA recovery.
Elution Procedure
• Ensure the ZymoBIOMICS™ DNase/RNase Free Water
hydrates the matrix for at least 1 minute before centrifugation.
• To increase yields, heat the ZymoBIOMICS™ DNase/RNase
Free Water to 60ºC before use. Additionally, users can reload
the eluate onto the column matrix, incubate at room
temperature for 3 minutes, and centrifuge again to increase
yield without further dilution.

Page 14
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Ordering Information
Product Description
Catalog No.
Kit Size
ZymoBIOMICS™ DNA Microprep Kit
D4301
50 preps.
ZymoBIOMICS™ DNA Microprep Kit (Lysis Matrix Not Included)
D4305
50 preps.
ZymoBIOMICS™ DNA Miniprep Kit
D4300
50 preps.
ZymoBIOMICS™ DNA Miniprep Kit (Lysis Matrix Not Included)
D4304
50 preps.
ZymoBIOMICS™-96 DNA Kit (Includes BashingBead™ Lysis Rack)
D4303
2x96 preps.
ZymoBIOMICS™-96 DNA Kit (Includes BashingBead™ Lysis Tubes)
D4309
2x96 preps.
ZymoBIOMICS™-96 DNA Kit (Lysis Matrix Not Included)
D4307
2x96 preps.
ZymoBIOMICS™-96 Magbead DNA Kit (Includes BashingBead™ Lysis Rack)
D4302
2x96 preps.
ZymoBIOMICS™-96 Magbead DNA Kit (Includes BashingBead™ Lysis Tubes)
D4308
2x96 preps.
ZymoBIOMICS™-96 Magbead DNA Kit (Lysis Matrix Not Included)
D4306
2x96 preps.
ZymoBIOMICS™, Zymo-Spin™, and DNA/RNA Shield™ are trademarks of Zymo Research Corp.
Disruptor Genie® is a registered trademark of Scientific Industries, Inc. FastPrep® is a registered trademark of Qbiogene, Inc. Illumnia® MiSeq™, Illumnia® NexTera® XT are trademarks or registered trademarks of Illumina Inc. AcroMetrix™ is a
trademark of Thermo Fisher Scientific Inc. ZymoBIOMICS™ DNA Miniprep Kit is for research use only. ZymoBIOMICS™ DNA Miniprep Kit is not sold for use in diagnostic procedures. Reagents included with this kit are irritants. Follow the
safety guidelines and rules enacted by your research institution or facility including the wearing of protective gloves and eye protection when using this kit.
For Individual Sale
Catalog No.
Amount
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm)
S6012-50
50
ZymoBIOMICS™ Lysis Solution
D4300-1-40
40 ml
ZymoBIOMICS™ DNA Binding Buffer
D4300-2-100
100 ml
ZymoBIOMICS™ DNA Wash Buffer 1
D4300-3-50
50 ml
ZymoBIOMICS™ DNA Wash Buffer 2
D4300-4-60
60 ml
ZymoBIOMICS™ DNase/RNase Free Water
D4302-5-10
10 ml
Zymo-Spin™ IICR Columns
C1078-50
50
Zymo-Spin ™ III-F Filters
C1057-50
50
OneStep™ PCR Inhibitor Removal Kit
D6030
50
Collection Tubes
C1001-50
C1001-500
C1001-1000
50
500
1,000
Sample Collection
Catalog No.
Amount
DNA/RNA Shield™ - Lysis Tube (Microbe)
R1103
50
DNA/RNA Shield™ – Fecal Collection Tube
R1101
10
DNA/RNA Shield™ – Swab and Collection Tube
R1106
R1107
10
50
DNA/RNA Shield™
R1100-50
R1100-250
50 ml
250 ml