eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Basic protocol to split Human Embryonic Kidney 293 (HEK293) cells to maintain them throughout the week and move on to cell culture plating with leftover cells for future experiments.</p>

[BEFORE_START]
<p style="text-align: left;">Make complete DMEM:</p>
<p style="text-align: center;"> </p>
<table style="height: 105px;" width="312">
<tbody>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;"><strong><em>Reagent</em></strong></td>
<td style="width: 148px;"><strong><em>Volume</em></strong></td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">DMEM</td>
<td style="width: 148px; text-align: center;">432.5 mL</td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">FBS</td>
<td style="width: 148px; text-align: center;">50 mL</td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">Pen/Strep</td>
<td style="width: 148px; text-align: center;">5 mL</td>
</tr>
<tr>
<td style="width: 148px; text-align: center;">HEPES (1M, pH 7.4)</td>
<td style="width: 148px; text-align: center;">12.5 mL</td>
</tr>
</tbody>
</table>
<p> </p>

[STEPS]
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TITLE: The calculation of gut metabolic modules form gene profile
AUTHORS: Qi Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The calculation of gut metabolic modules form gene profile</div></div>

[STEPS]
?. Step 1: the putative amino acid sequences were translated from the gene catalogues and aligned against the proteins/domains in the KEGG databases (release 79.0, with animal and plant genes removed) using BLASTP (v2.2.26, default parameter except that -e 0.01 -b 100 -K 1 -F T -m 8). Each protein was assigned to the KO group by the highest scoring annotated hit(s) containing at least one HSP scoring >60 bits.The relative abundance profile of KOs was determined by summing the relative abundance of genes from each KO.
?. Step 2: The calculation of gut metabolic modules by one command:'java -jar gmms.jar -a 2 -d GMMs.v1.07.txt -i input_KO_profile -s average -o index'You could refer to the http://www.raeslab.org/companion/gmms/ for 'gmms.jar' and 'GMMs.v1.07.txt'.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides methods for quality control of metagenomic data. Included is adapter trimming, quality trimming, decontamination, negative control removal, and pre-processing results. Based on the methods found in the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplementary information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software:<br /></strong>
<ul>
<li>cutadapt-1.4.1</li>
</ul>
<ul>
<li>fastx_toolkit-0.0.14</li>
<li>NCBI's BLAST+ v2.2.0</li>
<li>bwa-0.5.9</li>
</ul>
<ul>
<li>deconseq-standalone-0.4.3</li>
</ul>
<strong><br />Relevant Files<br /><br /></strong>Output:<br />
<ul>
<li>Virome_Sequence_Counts</li>
<li>Whole_Microbiome_Sequence_Counts</li>
</ul>
Perl Scripts:<br />
<ul>
<li>calculate_fasta_median_length.pl</li>
<li>calculate_fastq_median_length.pl</li>
</ul>
R Scripts:<br />
<ul>
<li><a href="https://www.protocols.io/view/Script-R1-Virome-Contig-and-Sequencing-Statistics-eh5bb86?step=undefined" target="_blank">R1</a>, <a href="https://www.protocols.io/view/Script-R2-Whole-Metagenome-Contig-and-Sequencing-S-eh6bb9e" target="_blank">R2</a>, and <a href="https://www.protocols.io/view/Script-R3-Quality-Control-Statistics-eh9bb96" target="_blank">R3</a></li>
</ul>
<ul>
<li>human_decontamination_stats.R</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocols is for RNA extraction from Whatman filter paper.

[STEPS]
SECTION: Lyse RBC
?. 
SECTION: Precipitate the DNA and separate phases
?. 
SECTION: Determine the DNA yield
?. 
SECTION: Precipitate the DNA and separate phases
?. 
SECTION: Precipitate the DNA and separate phases
?. 
SECTION: Precipitate the DNA and separate phases
?. 
SECTION: Lyse RBC
?. 
SECTION: Lyse RBC
?. 
SECTION: Precipitate the DNA and separate phases
?.
TITLE: Knockout PCR
AUTHORS: Brian    Teague

[DESCRIPTION]
The polymerase chaine reaction (PCR) amplifies linear DNA using a DNA polymerase enzyme and a pair of short single-stranded DNA "primers." This protocol amplifies a region that spans the Cas9 cut site so you can verify whether or not the URA3 "patch" inserted correctly (and thus disabled your gene.)


  


[STEPS]
1. Check that the thermocycler is programmed with the following program and holding at 98 °C



 


 


2. Grab an ice bucket and fill it with ice. If you don't have an ice bucket, a beaker will work in a pinch.
3. If necessary, dilute the primers to a concentration of 10 micromolar (µM) in  . (Make 100 µL of each dilution.)

 

4. Mix the following in a PCR tube on ice, in this order:
7 µL  
1 µL forward primer
1 µLreverse primer
1 µL template DNA
10 µL  





5. Mix the reaction by gently flicking the tube several times, then spin down in the microfuge.

 

6. Transfer the tube from ice to a pre-heated thermocycler holding at 98 °C. Start the PCR program.


7. After the PCR program has run, store the tube at-20 °C
TITLE: Project YES! Youth Engaging for Success: Arthur Davison Children's Hospital Pathology Laboratory's Standard Operating Procedures for HIV viral load and resistance testing
AUTHORS: Julie Denison, Jonathan Mwansa, Sam Miti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Arthur Davison Children's Hospital Pathology Laboratory’s Standard Operating Procedures used to test Project YES! (Youth Engaging for Success) HIV viral load and resistance testing.  </div></div>

[STEPS]
TITLE: Flow Cytometry ICS Nuclear Antigens
AUTHORS: Michael Betts, Gregory Golden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. A combined analysis of extracellular epitope staining (ECS) and intra-cellular epitope staining (ICS) using a collection of fluorophore-labeled antibodies sufficiently identifies discrete cell populations and their respective phenotypes. Importantly, ECS/ICS can be applied to cryo-preserved cell suspensions recovered in tissue culture media, enabling samples to be conveniently analyzed after collection and proper cryopreservation. However, consistent cryopreserved sample recovery and ECS procedures are critical to data comparison across multiple experiments. Herein, we describe a standardized protocol for cryopreserved sample recovery and ECS/ICS procedures for cell-surface and intra-cellular epitope labeling.</div></div>

[STEPS]
?. [Procedure]
Thawing and Restinga. Pre-warm R10 media in a  water bath. b. Thaw samples in-vial using a  water bath.c. Add thawed cells to  of R10, then spin cells at 500 xg for 5 min.d. Resuspend cell pellet in of R10 and count cells. e. Rest cells at least 3 hours (up to overnight) at 2x106 cells/mL in R10 medium + 1 µL/mL DNAse I at  , 5% CO².Note: during resting, prepare antibody cocktail master mix. Adjust volume of ECS for  per test with FACS buffer.f. After resting, add PBS up to  or  (whichever is closer, rounding up) to cells and transfer to  or  conical tube.g. Spin cells at 500 xg for 5 minutes at room temperature (RT).h. Resuspend cells in PBS to 1x107 cells/mL and count. If cells are too dilute, re-spin cells and resuspend at 1x107 cells/mL. Transfer  of cells (2x106 cells) into each well of a V-bottom 96 well plate.
37 °C
37 °C
14 mL
3 mL
37 °C
50 µl
15 mL
50 mL
15 mL
50 mL
200 mL
?. [Procedure]
Viability and extracellular staining (ECS)a. Spin plate at 500 xg for 5 minutes at RTb. Using a multichannel pipette, carefully remove the supernatant. c. Add of 1:60 Aqua viability dye directly to cell pellet and resuspend cells.d. Incubate for 10 minutes at RT in the dark.e. Add of ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).f. Add  of FACS buffer to each well and spin plate at 500 xg for 5 minutes. Remove supernatant.
5 µl
50 µl
100 µl
?. [Procedure]
Fixation, Permeabilization, and ICS Staininga. Fix cells with  of 1xFixation/permeabilization buffer (1 part concentrate + 3 parts diluent) for 30 minutes at RT in the dark.Note: Make ICS abs hereb. Add of 1x perm/wash buffer and centrifuge 800 xg for 5 min.c. Remove supernatant and add ICS cocktail (made in perm/wash buffer, made with diH²O) to the cells.d. Incubate for 1h at RT in the dark.e. Add  perm/wash buffer. Centrifuge at 800 xg for 5 min.f. Discard supernatant and resuspend pellet in 1% PFA and transfer to FACS tubes.g. Wrap in aluminum foil and store at  until flow cytometry.
100 µl
100 µl
100 µl
100 µl
200 µl
4 °C
TITLE: Growth Curve Stress Test
AUTHORS: Brian    Teague

[DESCRIPTION]
This is one (fairly quantitative and reproducible) way to run a yeast growth study to analyze the effect of a stressor on yeast. It is not the only way! It is also less of a "protocol" and more of a "template" -- it measures the growth of a single yeast strain at one concentration of "chemical X." Makes sure to adapt it to your own needs!

[GUIDELINES]
Remember, if you see a difference between the knockout and the wild-type under stress, it could be that the stress is slowing down the knockout strain. However, there's another possible explanation: it could just be that the knockout strain is growing more slowly under all conditions. Hence the no-stress controls.

[STEPS]
SECTION: Assay setup
1. 
 

Vortex the yeast culture briefly to resuspend the yeast cells.

SECTION: Assay setup
2. Set up four wells according to the following table:
 
Well 1Well 2Yeast culture50 µl50 µlChemical X, 100 mM10 µl--Water40 µl50 µl

SECTION: Assay setup
3. Set up the plate reader as follows:
Temperature: 30°C
Mode: Kinetic
Wavelength: 600 nm
Interval: 5 minutes
Total run time: 24 hours
Shake before read: 30 seconds
SECTION: Assay setup
4. Transfer the assay plate to the reader and read for 24 hours.
SECTION: Data analysis
5. Plot each growth curve with the Y axis scale set to "logarithmic."
  

SECTION: Data analysis
6. Find the part of the curve that is linear.  
 

SECTION: Data analysis
7. Find the OD600 reading at these two times. 
 

SECTION: Data analysis
8. Compute the doubling time using a bit of math:
  
Where t1 and t2 are the start and end times you found in step 2, and q1 and q2 are the OD600 at those times.
TITLE: i-mJOA Methodology
AUTHORS: Bryn Hilton, Jennifer Tempest-Mitchell, Benjamin Davies, Mark Kotter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was used to retrospectively infer modified Japanese Orthopaedic Association (mJOA) scores in a study conducted in 2016-2017 investigating various features of the diagnostic pathway patients experiencine in Degenerative Cervical Myelopathy (DCM). </div></div>

[STEPS]
?. [Methodology]
Introduction
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							The modified Japanese Orthopaedic Association (mJOA) scoring system is well-validated tool used for grading the severity of degenerative cervical myelopathy through a patient questionaire examining 4 domians: upper limb dysfunction, lower limb dysfunction, sensory dysfunction, and urinary dysfunction. For our study, we developed an inferred mJOA scoring system (i-mJOA) that could be used to estimate true mJOA within a small degree of error without having true mJOA scores available. The aim of i-mJOA was to facilitate retrospective disease severity assessment in our centre. It is not intended for general use as a research tool more broadly and would require extensive cross-validation in order to be applied in any other context.
?. [Methodology]
Results: Kappa statistical comparison:Total mJOA vs. Total i-mJOA: 0.73Upper limb mJOA vs. Upper limb i-mJOA: 0.66Lower limb mJOA vs. Lower limb i-mJOA: 0.75Sensory mJOA vs. Sensory  i-mJOA: 0.32Urinary mJOA vs. Urinary i-mJOA: 0.29Average differnce in category scores (mean +/- standard deviation):Total mJOA vs. Total i-mJOA: 0.13±1.51Upper limb mJOA vs. Upper limb i-mJOA: 0.13±0.92Lower limb mJOA vs. Lower limb i-mJOA: 0.13±0.92Sensory mJOA vs. Sensory  i-mJOA: -0.4±0.74Urinary mJOA vs. Urinary i-mJOA: 0.27±0.70
?. [Methodology]
Methodology:1. We developed a conversion chart that translated details in patients' clinical records into mJOA subcategory scores. (Table 1)2. The true mJOA scores of 13 patients in our centre were collected and their clinical records were anonymised. These patients were outside the time frame used for the study we later applied i-mJOA to and thus did not introduce bias. 3. The mJOA scores of these patients were removed from their records.4. We applied our conversion criteria to these patients' notes. 5. mJOA and i-mJOA were statisticaly analysed using the kappa statistic. Results below.
?. [Methodology]
TITLE: Flow cytometry
AUTHORS: Pranay Srivastava

[DESCRIPTION]
This protocol is to assess immune cell profile inn spleen (Chauhan et al. 2018). 

[STEPS]
1. Spleen was removed in a 35 mm petri plate with 5 ml RPMI 1640 and digested mechanically and passed through 70 μm filter screen. 
2. The cell suspension was centrifuged, and the pellet was incubated in RBC lysis buffer. The resulting cell suspension was washed in 1xPBS and blocked with Fc Block (Biolegend, Cat# 101302, 1 μl/50 μl).

3. The cells were incubated with MC1R (Invitrogen, Cat# PIPA521911, 1.39 µg) antibody followed by fluorophore conjugated primary antibodies for extracellular markers (Biolegend Cat# 101235,
CD11b-BV421(0.25 µg); Cat# 127641, Ly6G-BV-650 (0.25 µg); Cat# 128041, Ly6C-BV785 (0.125 µg); Cat# 100516, CD4-APC (0.25 µg); Cat# 100751, CD8a-BV510 (0.5 µg); Cat# 152405, CD19-PerCP-Cy5.5 (0.25 µg); Cat# 102036, CD25-BV605 (0.3 µg)) and AF488 (Invitrogen, Cat# A11034, 1:200). 
4. Zombie dye (Biolegend, Cat# 423101, 1µl/sample) was used to differentiate between live and dead cells. Helper T cells and cytotoxic T cells were identified by CD4+ and CD8+, respectively.
CD4+CD25+ cells were used to mark Tregs. CD19+ cells were used as marker for B cells. Monocytes were identified as CD11b+Ly6G-Ly6Chigh cells and neutrophils were marked by CD11b+Ly6C-Ly6G+. For monocytes and neutrophils, CD11b-positive cells were first extracted from the live cell subset by
expansion with SSC-A, followed by Ly6C. After expansion with Ly6C and Ly6G, we excluded Ly6G-positive cells. Monocytes were identified as CD11b+Ly6G-Ly6C high cells, and neutrophils were marked by CD11b+Ly6C-Ly6G+. 
5. SORP 5 Laser BD Fortessa X-20 (BD Bioscience) and FlowJo v10.7.1 (Becton Dickson & Company) software was used for data acquisition.
TITLE: Measurement of bacterial dry weight
AUTHORS: Alfonso   Pérez Escudero, Gabriel Madirolas

[DESCRIPTION]
Protocol to measure the biomass content of bacterial cultures, by measuring their dry weight after evaporation of water.

[STEPS]
1. Inoculate one or two colonies of bacteria into 5 mL LB in a closed 50-mL Falcon tube. Incubate with orbital shaking   

2. Inoculate 20 microliters of the culture prepared in step #1 into 200 mL of LB, in a 1 L flask. Cover flask with aluminium foil, and incubate for 24 hours with orbital shaking   

4. Wash culture resulting from step #2 in M9 worm buffer 2 times. For each wash: Transfer the culture to 50-mL Falcon tubes, centrifuge 5000 x g, 5 min, 4 °C, remove supernatant and resuspend pellet in M9 worm buffer. After the last wash, resuspend in M9 worm buffer up to 5.5 mL.
5. Measure optical density of the resulting bacterial suspension.
6. Add 5 mL of each bacterial suspension to each glass vial. Also, prepare an extra glass vial with 5 mL of M9 worm buffer.
7. Leave glass vials with bacterial suspension and M9 in an oven at 90 °C  for 1440 min

Note: In our experience, 24 hours are enough for all water to evaporate completely. You can check this by repeating step # 8 at different times, until the weights stabilize.
8. Weigh again the glass vials, which now contain the solid residue from the bacterial cultures. The weight of the vials may increase slightly over time from the moment you take them from the oven, as they acquire humidity from the air. To control for this as much as possible, take them out one by one, and wait for a fixed amount of time so that they cool down before weighing (usually 1 minute is enough).
9. Compute density of dry weight at OD=1 as ((wb - wbt) - (wm - wmt))/(OD*V),

where
- wb is the weight of the tube with the dried bacterial residue (measured in step #8)
- wbt is the weight of the same tube, but clean (measured in step #3)
- wm is the weight of the tube with the dried M9 residue (measured in step #8)
- wmt is the weight of the same tube, but clean (measured in step #3)
- OD is the optical density of the bacterial suspension (measured in step #5)
- V is the volume that was added to the vials in step #6 (5 mL)
3. Take clean glass vials, and put them into an oven at 90 C for at least 2 hours, in order to ensure that they are completely dry. Then, weigh each vial separately in a high-accuracy scale (resolution of 0.0001 g or better)
TITLE: U Mass - Surgery – carotid artery cannulation
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Chronic indwelling catheter is placed in the carotid artery for blood sampling during experiments. Surival surgery is performed in anesthetized mice, and mice recover from surgery after 4~5 days.</div></div>

[STEPS]
?. Anesthetize mice with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight).
?. Check the level of general anesthesia using a pinch stimulus of animal's tail and/or foot.
?. Make a transverse incision (~0.5 cm) over the trachea, and isolate the carotid artery.
?. Carefully introduce a silastic catheter (PE 10) into the vessel. The catheter should be filled with a saline solution containing heparin (10 U/ml) and plugged.
?. Guide the catheter to the back of the neck, and place under the back skin to prevent its accessibility from the mouse.
?. Tie a silk to the catheter, and make a small opening at the back of neck.
?. This silk, which is partially exposed, will be used/pulled on the day of experiment to expose the catheter.
?. On the day of experiment, flush the catheter using a heparinised saline solution.
?. All surgical procedures should be performed using standardized aseptic techniques, and all surgical tools should be autoclaved following the surgery.
?. Apply post-operative care based on the institution’s animal care and use committee standard operating policy.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> </p>
<p> </p>
<p>We are still developing and optimizing these protocols!</p>
<p> </p>
<p>The aquatic vertebrate eDNA protocols are  designed for persons familiar with basic molecular biology techniques and access to essential molecular biology laboratory equipment. To facilitate use, we utilize commercial kits and open source software, and standardized PCR and sequencing protocols.</p>
<p> </p>
<p><strong>Overview. </strong>From start to finish there are four laboratory components, each of which can potentially be completed in one day. Except for filtration, which we aim to do within 0-2 days of collection, samples can be safely stored for weeks to months between steps at -20 °C. All procedures are done on an open bench wearing gloves; wipe down work area and equipment after use with 10% bleach. We change gloves a lot to minimize cross contamination. Separate sets of pipettors and reagents are used for pre- and post-PCR work. We do one component at a time; in particular when filtering do not perform other procedures including DNA isolation and PCR. Unfiltered pipette tips are employed; after each procedure used tips are discarded and collection containers rinsed with 10% bleach. To reduce potential for cross-contamination, we do PCR in GE Illustra 8-tube strips (i.e., not 96-well plates), and other steps in individual 1.5 ml microfuge tubes.</p>
<p> </p>
<p>LABORATORY COMPONENTS</p>
<p> </p>
<p>1. Filter water samples</p>
<p>2. Isolate DNA</p>
<p>3. Amplify vertebrate 12S</p>
<p>4. Add Nextera tags</p>
<p> </p>

[STEPS]
?.
TITLE: In vitro LRRK2 kinase activity assay using mass-spectrometry as readout
AUTHORS: Verena Dederer, Deep Chatterjee, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine in vitro substrate phosphorylation for any purified kinase substrate pair.

It can be used to determine inhibition curves by addition of concentration series of kinase inhibitors.

Reaction is carried out in 50 µL reaction mix containing final concentration of 50 nM kinase and 5 µM substrate.

[STEPS]
6. Incubate reaction for 3 h at room temperature.
Note: depeding on kinase this may be shorter or longer.
SECTION: Step-by-Step Protocol
1. Prepare 50 µL reaction mix I: 50 nM purified LRRK2RCKW and 5 µM Rab8 substrate in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.
Aliquot 25 µL per tube.


 

SECTION: Step-by-Step Protocol
3. Prepare 25 µL reaction mix III: same as reaction mix II but no ATP as negative control.
SECTION: Step-by-Step Protocol
4. Start reaction by adding 25 µL reaction II to reaction mix I.
Note: for negative control add 25 µL reaction mix III to second aliquot of reaction mix I.
SECTION: Step-by-Step Protocol
5. Mix by vortexing and brief centrifugation for 30 sec with 500xg.
SECTION: Step-by-Step Protocol
2. Prepare 25 µL reaction mix II: 2 mM ATP + 2.5 mM MgCl2 in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.

 

7. Stopp reaction by adding 50 µL mass spec buffer (dH2O+0.1% formic acid).
8. Store at -80°C or proceed directly with mass spectrometry analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To prepare the genomic DNA of microsporidia, 400 ll suspension of purified spores (1010 spores/ml) was mixed with 40 ll KOH (2 mol/l) in a 1.5 ml Eppendorf tube and incubated at 27C for 1 h, TEK buffer (1 mmol/l Tris–HCl, 10 mmol/l EDTA, 0.17 mol/l KCl, pH 8.0) was added and continued incubating at 27C for 1 h. Adjusted pH to 8.0 with 1 mol/l HCl, added 10% SDS in order to attain 0.5% in the mixture and kept it in ice-bath for 15 min. 20 mg/ml proteinase K (TaKaRa Biotechnology Co. LTD) was added in order to attain 200 lg/ml in the mixture and incubated at 50C for 4 h. Subsequently, isovolumetric tris–phenol extraction was performed twice, and washed with chloroform: isoamyl alcohol (24:1 v/v). 10% NaOAC was added to the recovered aqueous phase after centrifuging (10,000 r/min, 5 min), and then the DNA was precipitated by 2.5 times volume of cold ethanol at -20C for 40 min before centrifuging (12,000 r/min, 10 min). In addition, the precipitated DNA was rinsed twice with 500 ll cold 70% ethanol before drying at 37C for 5–10 min. Then, the extracted DNA was stored at -20C after dissolution in 50 ll TE buffer at 65C for 10 min. </p>

[STEPS]