SSU rDNA GeneChip Protocol 1/06/02

**[Outline]{.underline}**

**[A. Target Preparation]{.underline}**

1.  PCR amplification and quantitation of the 117 bp product used for
    target hybridization

2.  Fragmentation of 117 bp product

3.  Biotin labeling of fragmented product = target for GeneChip probe
    array

**[B. GeneChip Preparation]{.underline}**

1.  Prehybridization of GeneChip probe array

2.  Target hybridization to Gene Chip probe array

3.  Experiment/Fluidics station setup

4.  Washing and staining of GeneChip probe array

5.  Cleaning the fluidics station/ shutdown

**[C. GeneChip Analysis]{.underline}**

1.  Probe array scan

2.  Data Analysis

**[Appendix 1.]{.underline}** Environmental DNA isolation protocol

A.  **[Target Preparation]{.underline}**

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1.  **PCR Amplification of 117 bp product for target hybridization**

> Reaction Mix:
>
> **1X**
>
> Sterile dH2O (Teknova) 83 μl
>
> 10X Clontech AdvanTaq buffer 10
>
> 10mM (total) dNTP's 2
>
> 20μM Pc5B 1.5
>
> 20 μM cCompLong 1.5
>
> AdvanTaq (Clontech) [1]{.underline}

Total: 99 μl

Add 1 μl environmental DNA, 1 μl of a 1:10 dilution of full 16S
bacterial amplicon (positive control) or 1 μl sterile H~2~O (negative
control) to each tube for a final volume of 100 μl.

Thermoprogram:

95°C 1 min

95°C 15 sec

45°C 30 sec **28-35 cycles**

68°C 45 sec

68°C 10 min

4°C Hold

After thermoprogram is finished, run 7 μl of each sample and 2, 4 and 6
μl of low mass ladder on a 3% Nusieve agarose gel at 110V for 1 hour. We
quantitate our PCR products to determine the volume to add to the
fragmentation reaction, which cuts the products into 20-80 bp fragments.

We analyze gels using the FluorS-MultiImager (BioRad, Hercules, CA).

**Fluor-S MultiImager Operating Instructions**

After staining the gel in ethidium bromide, lift the lid of the scanner
and load the gel onto the middle of the glass surface.

On the attached computer, select Quantity One program, then open
Fluor-S. A screen will appear with three steps.

Step 1: Ethidium bromide, 520 LP, UV, Trans, HiRes and Scan Dim. 160 mm
should all be selected.

Step 2: Select position. Gel will appear on screen: position the gel to
the middle of the grid marks. Gel should already be focused.

Step 3: Set exposure time of 20 sec, the select acquire. The gel will
then be scanned into the computer.

Gel will appear on computer screen. Transform gel using the tool to
obtain clearer image of product bands. Zoom into desired bands and
standards using tool.

Take picture of gel: File\> Print\> Video Print

**DNA Mass Calculation: Open Volumes Quick Guide.**

Open volume contour tool in the volumes quick guide. The tool is
activated when you see a + symbol: put the + on the desired band: move
it around until it outlines the band with a green line. Continue
outlining all bands, including mass ladders and unknowns. Once all bands
are outlined, zoom back out ( tool).

Using the volume rectangle tool, pick an area representative of the
background near the unknown bands. Highlight the rectangle to be yellow:
double click for volume properties, fill in form to say background.

For standard bands: highlight each band yellow: double click: fill in
form: label as standard and fill in band mass concentrations (key is
taped onto computer screen).

For unknown bands: highlight each band yellow, label as unknown.

After all inputs are made, save the file.

Using volume regression curve tool, calculate the correlation
coefficient. We use anything above 70%. It's okay to remove any bands on
the dye line that don't fall around the curve (the background is
different there and these bands don't usually correlate well to the
others).

The volume analysis report tool lists the information generated.
Reformat the report so that index, name, and concentration only are
reported. Print report to default printer toto, located across the hall
in Wendy/Todd's office 1250.

The report shows the concentration of the product loaded on the gel:
since 7 μl was added to gel, divide by 7 to get the ng/μl concentration.

2.  **Fragmentation of the 117 bp product**

1\. **Prepare Fragmentation Working Mix:**

> a\. Calculate amount of Affymetrix fragmentation reagent to add:
>
> **for 6 rxns: 0.75** Units/ working mix ÷ concentration of
>
> fragmentation reagent (on tube) = 0.5X
>
> **for 12 rxns**: **1.5** Units/working mix ÷ conc of frag reagent =
> 0.5X
>
> **for 24 rxns**: **3** Units/working mix ÷ conc of frag reagent = 0.5X
>
> Example: 1.5 Units/ 1.7 Units/μl = 0.88 μl/ working mix = 0.5 X
>
> Add other working mix components according to the following chart:
>
> [Component \# rxns Volume (μl) Final Conc.]{.underline}
>
> Frag. rgt 1-5 0.5X 0.125 Units
>
> 6-12 0.5X 0.125 Units
>
> 13-24 0.5X 0.125 Units
>
> dH~2~0 same as frag rgt 0.5X
>
> EDTA (20mM) 1-5 X variable
>
> 6-12 X variable
>
> 13-24 X variable
>
> Alkaline phosphatase
>
> (1U/μl) 1-5 15 2.5 Units
>
> 6-12 30 2.5 Units
>
> 13-24 60 2.5 Units
>
> Tris-acetate
>
> (10 mM) 1-5 15-(2)X μl 0.5 mM
>
> 6-12 30-(2)X μl 0.5 mM
>
> 13-24 60-(2)X μl 0.5 mM
>
> **Final Volumes of Working Mix:**
>
> 1-5 rxns: 30 μl

12. rxns: 60 μl

> 13-24 rxns: 120 μl
>
> 2\. From gel electrophoresis results, **calculate amount of PCR
> product** to add for a concentration of 500 ng DNA. To a PCR tube add:
>
> 117 bp product (500 ng total): Formula: 500 ng ÷ ng amt/μl from gel
> quantitation = product μl
>
> Sterile dH~2~O: 45 μl total rxn volume -- product μl = μl water to
> add.
>
> 3\. **Fragmentation Reaction**: Add 5 μl fragmentation working mix to
> the [side of tube]{.underline}. (It's important that all reactions are
> started at the same time.) After working mix is added to each tube,
> and thermocycler is ready, knock working mix into liquid and vortex
> briefly. Immediately place in 9600 thermocycler using the following
> program:

25°C 12 min

95°C 10 min

4°C Hold

> Optional: Run 7 μl of each fragmented DNA sample and a 25 bp standard
> on a 3% NuSieve Gel at 115 V for 55 min. Expected results: smear of
> DNA between 25-100 bp markers. If smear is not present, or DNA is not
> cut uniformly; do not proceed.

3.  **Biotin Labeling of fragmented product = target for GeneChip probe
    array.**

> 1\. **Biotin Reaction Mix**: (use PCR tubes)
>
> **1X**
>
> 5X TdT buffer (BM) 7 μl
>
> 25 mM CoCl~2~ 2.5 μl
>
> TdT Enzyme 25U/μl 2 μl
>
> Biotin-N6-ddATP (1 mM) 2 μl
>
> Fragmented product 21.5 μl
>
> Total Volume 35 μl
>
> 2\. **Biotin Labeling Reaction**: Use 9600 thermocycler to run
> reaction:
>
> 37°C 2 hrs
>
> 4°C Hold

B.  **[GeneChip Preparation]{.underline}**

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1.  Prehybridization

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a.  Equilibrate genechip probe array to room temperature

b.  "Prewet" array: Insert an unfiltered pipet tip in one of the
    genechip septa to vent air from chamber. Then load hybridization
    buffer (35 μl 5M TMAC + 185 μl Rapid Hyb Buffer (Amersham)) through
    the other septum.

c.  Incubate the genechip in the hybridization oven at 45°C and 60 rpm
    for at least 10 min.

2\. Target preparation and hybridization to GeneChip probe array

a.  While genechip is equilibrating (in prehyb), prepare target
    cocktail:

> 35 μl 5M TMAC
>
> 185 μl Rapid Hyb Buffer
>
> 17 μl Biotin-labeled target sample
>
> [3 μl Biotin-labeled control oligo 213 (5nM)]{.underline}
>
> Final Volume: 241 μl

b\. Heat target cocktail samples in a heat block at 99°C for 5 min.

> c\. Incubate samples in a 45°C water bath for 5 min., then spin
> samples briefly.

d.  Retrieve genechip array and remove hybridization buffer from the
    assay cartridge; fill with the target cocktail samples.

e.  Place probe arrays in rotisserie oven so they are balanced around
    the axis. Rotate at 60 rpm, 45 °C for a minimum of 3 hrs.

3\. Experiment/Fluidics Station Setup

a.  **Experiment Setup.** Define, or name your experiment: Launch the
    software from the workstation and choose **Experiment Info** from
    the **Run** menu. The **Experiment Information** dialog box will
    appear; fill in the following parameters:

> [Experiment Name]{.underline}: (include date and sample name)
>
> [Probe array type]{.underline}: 16S RNA I = 1210_s66 (sense)
>
> [Lot \#]{.underline}: Expiration date of chip
>
> [Operator Name]{.underline}: Your name!
>
> [Sample type]{.underline}: Amplicon (target) description
>
> [Description]{.underline}: Organism name (or experiment name)
>
> [Project]{.underline}: Project name
>
> [Comments]{.underline}: Hybridization time in oven
>
> **Save** experiment information
>
> The Affymetrix software will use this information to process the
> experiment data; the data files generated will correspond to the
> experiment name.

b.  **Fluidics station setup.** Choose Fluidics from the Run menu. The
    Fluidics Station dialog box will appear. Select your experiment name
    from the drop-down list.

> i\. Choose Protocol in the fluidics station dialog box. From the
> drop-down menu, choose Prime.
>
> ii\. In reservoir A add 1X SSPE/ 0.005% TritonX-100. In reservoir B
> add 6X SSPE/0.005% TritonX-100. Put fresh water in water reservoir.

iii. To begin priming, Click Run for each fluidic module to be used..

4\. Washing and Staining the Probe Array.

a.  **Stain preparation.** Streptavidin-phycoerthythin (SAPE) stain is
    light sensitive; store in foil at 4°C. Do not freeze concentrated or
    diluted SAPE. Prepare fresh SAPE stain solution just before use.

> i\. Get stain solution ready (mix in 1.5 ml tube):
>
> 500 μl 6X SSPE/0.005% Triton X-100
>
> 25 μl 20 mg/ml acetylated BSA
>
> [3 μl Streptavidin-Phycoerythrin (SAPE)]{.underline}
>
> Total: 528 μl

ii. Put 1.5 ml tube with SAPE into the sample holder on the fluidics
    station module, making sure that the metal sampling needle is in the
    tube with its tip near the bottom.

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b.  **Probe array preparation**.

> i\. After at least 3 hours of hybridization, take the
>
> target solution out of chip → store at --20°C in a sterile
> microcentrifuge tube. (Be sure to vent chip while draining fluid)

ii. Fill the probe array with 250 μl of 6X SSPE/0.005% Triton-X 100.

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c.  **Fluidics station operation.** For each probe array to be
    processed, prepare a module on the fluidics station.

> i\. In the fluidics station dialog box on the computer screen, select
> the experiment name entered from the drop-down list.

ii. In the protocol drop-down list, select **Affystain**.

> The Affystain protocol is:
>
> 4 washes with 6X SSPE/0.005% TritonX-100 at 40°C;
>
> 2 Washes with 1X SSPE/0.005% TritonX-100 at 40°C, Stain 15 min at
> 40°C,
>
> 3 Washes 6X SSPE/0.005%TritonX-100 at 25°C.
>
> iii\. Choose **Run** in the fluidics station dialog box to begin the
> washing and staining. Follow directions on each module's LCD window
> for guidance.
>
> iv\. Insert selected probe array into the sample holder of a fluidics
> module while the probe array lever is the **Eject** position.

iii. The fluidics module LCR will display the progress of the washing
     and staining process. Refer to Troubleshooting section of this
     manual if problems occur. When the procedure is complete, the LCD
     will display **EJECT CARTRIDGE**. Move the cartridge holder lever
     to the eject position. After removing the cartridge, return the
     cartridge holder to the engage position. The module will run a wash
     automatically and will be ready to process a new chip.

iv. Remove the SAPE stain vial and replace with an empty microcentrifuge
    tube.

v.  Check the probe array for large bubbles (see troubleshooting section
    if there's bubbles). If none are present, probe array is ready to be
    scanned immediately after protocol is finished. Store chip in foil
    at 4°C if more than 30 minutes elapse before scanning.

**5. Cleaning the Fluidics Station**

a\. After the last chip is processed, take tubes out of the buffer
solutions A and B and

place in the water container. Select SHUTDOWN from the protocol
drop-down list

on the computer screen; let the protocol run to completion, then turn
off the station.

C.  **[GeneChip Analysis]{.underline}**

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1.  Probe Array Scan.

> a\. The following is from the Affymetrix Expression Analysis Technical
> Manual, revision 1, 1998.

2\. Data Analysis

> a\. From the Affymetrix Expression Technical Manual;
>
> **[Reagents and Materials]{.underline}**

A.  Target Preparation

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1.  PCR Primers:

> 117 bp product:
>
> cCompLong: 5' TTGTACACACCGCCCGTCA 3'
>
> Tm=60°C
>
> Pc5B: 5' TACCTTGTTACGACTT 3' Tm=44°C
>
> Full 16S product (optional):
>
> PoRev: 5' AGAGTTTGATCMTGG 3' Tm=43°C
>
> Pc5B: 5' TACCTTGTTACGACTT 3' Tm=44°C
>
> Order primers from *Genosys* on **BBRP oligo ordering web page**→
> http://bbrp-r.llnl.gov/oligo-pbin/oligo_order_form name: oligo
>
> password: primer

2.  PCR Enzymes, Reagents

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a.  **Water**: PCR certified, sterile, 500 mls; catalog \# 0227;
    *Teknova*

b.  **dNTP's**: catalog \# N808-0260(premixed) or #N808-0007; *Applied
    Biosystems*

c.  **Taq polymerase**:

> [117 bp product]{.underline}:

a.  **Advantage 2 Polymerase Mix**, Use 10X PCR buffer provided with
    enzyme, catalog \# 8430-2; *Clontech*

> [Full 16S]{.underline}:
>
> a\. AmpliTaq Gold Polymerase, use 10X PCR buffer
>
> w/MgCl~2~ provided with enzmye, catalog \# N808-0241;
>
> *Applied Biosystems*

3.  Electrophoresis

> **a. Ethidium Bromide**, 10 ml (10mg/ml), catalog \# 15585-011,
>
> *Invitrogen Life Technologies*
>
> [117 bp product]{.underline}:
>
> a\. **NuSieve 3:1 Agarose**, catalog \# 50090; *Biowhittaker Molecular
> Applications-Cambrex*
>
> **b. 25 bp DNA ladder,** 50 μg, Catalog \# 10597011;
>
> *Invitrogen Life Technologies*

**c. Low Mass DNA ladder,** 200 ul, Catalog \# 10068013;

> *Invitrogen Life Technologies*
>
> [Full 16S]{.underline}:
>
> **a.** **Ultrapure agarose**, electrophoresis grade, catalog \#
> 5510UA;
>
> *Invitrogen Life Technologies*
>
> **b. 100 bp DNA ladder,** 50 μg, Catalog \# 15628-019;
>
> *Invitrogen Life Technologies (product discontinued)*

B.  Fragmentation

> 1\. **GeneChip Fragmentation Reagent**, \[DNaseI in 10mM
>
> Tris-HCl (pH 7.5), 10mM CaCl~2,~ 10 mM MgCl~2~ , and 50% glycerol\],
> catalog \# 900131 (can't order separately from kit as of 6/00; must
> find supplier who will put DnaseI in this buffer system); *Affymetrix*
>
> 2\. **0.5 M EDTA**, catalog \# 15575020
>
> *Invitrogen Life Technologies*
>
> 3\. **400 mM Tris Acetate**, pH 8.2 catalog \# 0279-IL-E
>
> *Teknova*
>
> 4\. **Calf Intestine Alkaline Phosphatase**, 1Unit/μl, 1000U (CIAP),
> Catalog \# 18009-027
>
> *Invitrogen Life Technologies (product discontinued)*
>
> 5\. **Water**, PCR certified, 500 mls, catalog \# 0227; *Teknova*

C.  Biotin Labeling Target DNA

> 1\. **Terminal transferase (TdT)** with 5X reaction buffer and CoCl~2~
> solution, 500 U= 1 pack, Catalog \# 0220582
>
> *Roche Applied Science*

1.  **Renaissance Biotin-N6-ddATP**, 25 μl; Catalog

> \# NEL 508; *Perkin Elmer Life Sciences*

2.  **Water**, PCR certified, 500 mls; catalog \# 0227

> *Teknova*

D. Hybridization, Washing and Staining of Target

1.  **20X SSPE** (3M NaCl, 0.2M NaH~2~P0~4~, 0.02M EDTA),

> l liter; Catalog \# 16-010Y; *BioWhittaker-Cambrex*

2.  **Water**, Molecular Biology Grade, 1 liter;

> Catalog \# 16-001Y; *BioWhittaker-Cambrex*

3.  **Triton X-100**, catalog \# T-9284, *Sigma-Aldrich*

4.  **Control oligos**:

> i\. **Control oligo 213=Oligo B1** (Affymetrix HIV PRT Assay); biotin
> label=7 (sense chips):
>
> 5' biotin-7 CTG AAC GGT AGC ATC TTG AC 3',
>
> ii.**Control oligo 948** (antisense chips):
>
> 5' biotin-7 GTC AAG ATC GTA CCG TTC AG 3',
>
> Both oligos HPLC purified, 0.05 μmol.; order through the BBRP oligo
> ordering web page, from *Genosys*

5.  **Acetylated Bovine Serum Albumin** (BSA) solution;

> Catalog \# 15561-020; *Invitrogen Life Technologies*

6.  **GeneChip probe arrays**:

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i.  16SRNA I Sense, catalog \# 500364;

ii. Env. Monitor II Sense, catalog #500424

> **Other GeneChip arrays available:**

iii. 16SRNA I Antisense, catalog \# 500372;

iv. Env. Monitor II Antisense, cat \# 500425;

> All from *Affymetrix*

7.  **Streptavidin-Phycoerythrin (SAPE)**, catalog \# S866,

> *Molecular Probes, Inc.*

8.  **Tubing** (for fluidics station), peroxide silicone, tube #14,
    (16mm 1/16"), catalog \# 910-0016-016; *Watson Marlow*

9.  **Wash buffers**: please see Regent/Buffer Preparation section

10. **Hybridization Buffer:**

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1.  Rapid Hyb Buffer, 125 mls;

> Catalog \# RPN1635;
>
> *Amersham Biosciences Corp.*
>
> 2\. TMAC (Tetramethylammonium chloride),
>
> 500 mls; Catalog \# T-3411; *Sigma-Aldrich*

**[Reagent/Buffer Preparation]{.underline}**

1.  **20 mM EDTA**

> 100 μl 0.5 M EDTA (Invitrogen)

2.4 ml Molecular Biology grade H~2~O (BioWhittaker)

Total volume: 2.5 mls

Aliquot 100 μl of mixed solution to each of 25 500 μl tubes.

Store at room temperature.

**2. 10 mM Tris-Acetate pH 8.2**

9.75 ml Molecular biology grade water (BioWhittaker)

250 μ 400mM Tris-Acetate (Teknova)

Final Volume: 10 ml

Aliquot 500 μl of the mixed solution to each of 20 microcentrifuge
tubes.

Store at room temperature.

> **3. 10% Triton X-100**
>
> 90 mls Molecular grade water (BioWhittaker)
>
> 10 mls Triton X-100 (Sigma-Aldrich)

Final Volume: 100 mls

Mix thoroughly and store solution at room temperature.

> **4. Wash Buffer A: 1X SSPE/ 0.005% Triton X-100**

**100 mls 50 mls**

5 mls 20X SSPE (BioWhit) 2.5 mls

50 μl 10% Triton X-100 Stock Soln 25 μl 94.95 mls Mol. Biol. Grade H2O
(BioWhit) 47.48 mls

> Mix ingredients thoroughly and filter through a 0.2 μm or 0.45 μm
> vacuum filtration system. Store at room temperature in sterile vial.
>
> **5. Wash Buffer B: 6X SSPE/ 0.005% Triton X-100**

**100 mls 50 mls**

30 mls 20X SSPE 15 mls

69.95 mls Water (BioWhittaker) 34.98 mls

50 μl 10% Triton X-100 Stock 25 μl

> Mix ingredients thoroughly and filter through a 0.2 or 0.45 μm vacuum
> filter system. Store at room temperature in a sterile vial.

**[Vendor Companies]{.underline}**

Affymetrix, Inc.

3380 Central Expressway

Santa Clara, CA 95051

Phone: (888) DNA-CHIP (362-2447)

Fax: (408) 481-0435

**Reps:**

Kyle O'Connor (Technical Support)

> Phone: (408) 731-5654
>
> E-mail: Kyle_OConnor@affymetrix.com

Carsten Rosenow, Ph.D. (Staff Scientist)

Phone: (408) 731-5024

Fax: (408) 481-0422

Joe Hernandez (marketing)

Tarif Awad (past technical support)

Phone: (408) 731-5653

E-mail: Tarif_Awad@affymetrix.com

Mike Del Santo (Sales Administration Coordinator)

Phone: (408) 731-5949

Fax: (408) 481-944

Margaret Stonich (Regional Sales Manager)

Phone: (408) 358-3913

Jesse McGraw (Our LLNL Account Manager) \*\*\*\*

Phone: (831) 476-2092

Fax: (831) 476-4002

E-mail: jesse_mcgraw@affymetrix.com

Lance Waketa, Sales (408) 731-5690 \*\*\*\*very helpful

Janet Lankard (Regional Sales Manager)

Phone: (408) 731-5275

Connie Delvilla, Senior Credit Account Receivable Analyst (408) 731-5818

Jean Yi, accounts receivable, (408) 731-5974

Don Levine, Accounts: (408) 731-5629

Greg Colbert, Accounts: (408)-731-5487

Mella, Accounts: (408) 731-5089

**Service Contract contact: (we no longer have a service contract)**

Field Service Engineer (408) 731-5654 or 1-888-DNA CHIP

Florante (Preventive Maintenance Inspector), with Molecular Dynamics

Phone: (800) 800-4383 ext. 7415

Amersham Biosciences Corp.

800 Centennial Ave.

P.O. Box 1327

Piscataway, NJ 08855-1327

Phone: (800) 526-3593

Fax: (877) 295-8102

Web: http://www.apbiotech.com

Applied Biosystems - Applera

850 Lincoln Centre Dr.

Foster City, CA 94404

Phone: (800) 345-5800

Fax: (650) 638-5884

Web:
[http://www.appliedbiosystems.com](http://www.appliedbiosystems.com/)

BioWhittaker Molecular Applications - Cambrex

8830 Biggs Ford Rd.

Walkersville, MD 21793

Phone: (800) 638-8174

Fax: (301) 845-8338

For NuSieve agarose:

191 Tomaston St.

Rockland, ME 04841

Phone: (800) 341-1574

Fax: (207) 594-3491

Web for all Biowhittaker/Cambrex: <http://www.bmaproducts.com/>

Clontech Laboratories, Inc.- BD Biosciences

1020 East Meadow Circle

Palo Alto, CA 94303-4230

Phone: (800) 662-2566

Fax: (800) 424-1350

Web: [http://www.clontech.com](http://www.clontech.com/)

Invitrogen Life Technologies (Gibco BRL)

1600 Faraday Ave

P.O. Box 6482

Carlsbad, CA 92008

Phone: (888) 584-8929

Fax: (888) 584-8930

Web: [http://www.invitrogen.com](http://www.invitrogen.com/)

Molecular Probes, Inc.

4849 Pitchford Ave.

Eugene, OR 97402-9144

Phone: (541) 465-8300

Fax: (541) 344-6504

Web: [http://www.probes.com](http://www.probes.com/)

Perkin Elmer Life Sciences (NEN)

549-3 Albany St.

Boston, MA 02118

Phone: (800) 446-0035

Fax: (617) 482-1380

Web:
[http://lifescience.perkinelmer.com](http://lifescience.perkinelmer.com/)

Roche Applied Science

9115 Hague Rd.

P.O. Box 50414

Indianapolis, IN 46250-0414

Phone: (800) 262-1640

Fax: (800) 428-2883

Web: [http://biochem.roche.com](http://biochem.roche.com/)

Sigma-Aldrich

3500 Dekalb St.

St. Louis, MO 63118 USA

Phone: (800) 325-3010

Fax: (800) 325-5052 or (314) 771-5757

Web: [http://www.sigmaaldrich.com](http://www.sigmaaldrich.com/)

Teknova

355 Princeton Ave.

Half Moon Bay, CA 94019

Phone: (650) 728-2557

Fax: (650) 728-3160

Web: [http://www.teknova.com](http://www.teknova.com/)

Watson Marlow

37 Upton Technology Park

Wilmington, MA 01887-1018

Phone: (800) 928-7786

Fax: (978) 658-0041

Web: [http://www.watson-marlow.com](http://www.watson-marlow.com/)

**Appendix 1.**

**Standardized Protocol for Isolation of Environmental DNA using the
MoBio Soil DNA Isolation Kit**

1.  To the 2ml Bead Solution provided add 250 mg of soil sample

2.  Gently vortex to mix

3.  Add 60 ul of Solution S1, invert to mix

4.  Add 200 ul of Solution IRS (Inhibitor Removal Solution), invert to
    mix

5.  *A. Method 1:*

> Vortex tubes for 5 seconds, then place in 70°C water bath for 10
> minutes. Vortex for 10 seconds to prevent settling and place in water
> bath for 10 more minutes. Remove from bath, and place in Bead-Beater
> at 11,500 rpm (10,000 X g) for 3 minutes.

*B. Method 2:*

Shake tubes in Bio101 Fast Prep FP120 for 15 seconds at 4.5 m/s

6.  Centrifuge tubes at 10,000 x g for 30 seconds

7.  Transfer 450 μl supernatant to a clean microfuge tube (Some soil
    particles may transfer over with supernatant, which is acceptable)

8.  Add 250 ul of Solution S2, Vortex for 10 seconds, and incubate at 4C
    for 10 minutes

9.  Centrifuge tubes for 1 minute at 10,000 x g.

10. Avoiding pellet, transfer 450 ul of supernatant to a clean microfuge
    tube. There may be some debris floating at the top of the
    supernatant. Try to avoid transferring the debris as well.

11. Add 900 ul of Solution S3 and vortex for 5 seconds.

12. Load about 700 ul onto the provided spin filter, and centrifuge for
    1 minute at 10,000 X g. Discard flow through and apply remaining
    supernatant to filter. Centrifuge for 1 minute at the same speed.
    Discard flow through.

13. Add 300 ul of Solution S4 (Wash solution) to filter. Centrifuge for
    30 seconds at 10,000 X g,

14. Discard flow through

15. Spin for one minute to "dry" filter.

16. Place spin filter in new clean microfuge tube.

17. Add 50 ul of Solution S5 (elution solution) to the center of filter
    membrane

18. Centrifuge at 10,000 X g for 30 seconds.

19. Discard spin filter. The elute contains DNA.

NaOH washes:

20. Aliquot 200 μl of 0.4M NaOH in a MicroCon-100

21. Add DNA from step 19 and centrifuge 20 minutes at 500 X g (2,500
    rpm).

22. Refill the MicroCon with 400 μl of 0.4M NaOH.

23. Centrifuge 20 minutes at 500 X g; discard eluate. (2X)

24. Add 400 μl 10 mM Tris (pH 7.5) to neutralize

25. Centrifuge 20 minutes at 500 X g/ discard eluate

26. Add 50 μl 10 mM Tris (pH 7.5) and collect sample by inverting into
    tube; centrifuge for 2 minutes at 500 X g.

27. Store DNA at 4°C.