eamag/protocols-with-steps huggingface dataset:
TITLE: Mycology media
AUTHORS: Yin-Tse Huang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mycology media</div></div>

[STEPS]
?. WA+C agar Water agar + chloramphenicol (氯黴素 kill bacteria): As an environmental isolating mediaComponentAmountAgar20gChloramphenicol0.05gDI water1000 ml
ComponentAmountAgar20gChloramphenicol0.05gDI water1000 ml
?. PDA+C agarpotato dextrose agar + chloramphenicol (氯黴素 kill bacteria): Use when WA+C agar is not workingComponentAmountPDAper instruction on labelChloramphenicol0.05gDI water1000 ml
ComponentAmountPDAper instruction on labelChloramphenicol0.05gDI water1000 ml
?. Emerson YpSs Agar: For chytridsComponentAmountYeast extract 4g Soluble Starch15gK2HPO41gMgSO4 x 7H2O0.5gAgar20gDI water 1000ml
ComponentAmountYeast extract 4g Soluble Starch15gK2HPO41gMgSO4 x 7H2O0.5gAgar20gDI water 1000ml
TITLE: Cold-leaching extraction. A new methodology for obtaining inhibitory substances produced by bacteria in solid media.
AUTHORS: Catherine Cesa-Luna, Alberto Aguayo-Acosta, Antonino Baez, Jesús Muñoz-Rojas, Verónica Quintero-Hernández

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Inhibitory substances can be obtained and purified by different methodologies. Free cell supernatants obtained by centrifugation and filtration are commonly used for evaluating antimicrobial metabolites produced by bacteria. In this work, a new methodology for obtaining antimicrobial metabolites is proposed, especially implemented for bacteria which are not able to produce these compounds in liquid media; based on the use of nitrocellulose filter membranes and solid culture media.</div></div>

[STEPS]
TITLE: Experimental design
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
The protocol details the experimental design.

[STEPS]
SECTION: Experimental design
1. WT and G2019S mice were exposed to intraperitoneal (ip) injections of a low dose of lipopolysaccharide LPS (0,1 mg kg-1 Escherichia coli serotype O111:B4) (Sigma-Aldrich), administrated twice a week for 12 weeks (Supplementary Figure 1). Treatments were performed in two different age groups, 3M (young adult) and 7M (at the start of middle-age). WT and G2019S mice exposed to ip injections of 0.9% sterile NaCl were used as controls. 
SECTION: Experimental design
2. Young mice receiving the treatment for 12 weeks were sacrificed at 6 M; middle aged mice were subdivided in 2 groups; one group received the treatment for 12 weeks was killed at 10 M, the second group received the treatment for 12 weeks and from 10 M to 16 M received saline only (Supplementary Figure 1A-B).
SECTION: Experimental design
3. Mice (n = 20/experimental group) were randomly assigned to one of seven experimental conditions for each genotype: 
1. 3 M Basal (no injections); 
2. 6 M NaCl (NaCl injections started at 3 M); 
3. 6 M LPS (LPS injections started at 3 M); 
4.10 M NaCl (NaCl injections started at 7 M); 
5.10 M LPS (injections started at 7M); 
6. 16 M NaCl (injections started at 7 M); 
7.16 M LPS (injections started at 7 M). 
5. A second series of experiments were carried out (n=8 mice/experimental group) for immunohistochemical and protein analyses.
4. Clinical evaluation (body weight, mantel status, lethargy, reluctance to move, grooming behavior) was carried out weekly until sacrifice.
TITLE: UC Davis - HbA1c Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:   </span></div><div class = "text-block">Direct Enzymatic HbA1c test is an enzymatic assay in which lysed whole blood samples are subjected to extensive protease digestion with Bacillus sp protease. This process releases amino acids including glycated valines from the hemoglobin beta chains. Glycated valines then serve as substrates for specific recombinant fructosyl valine oxidase (FVO) enzyme, produced in E. coli. The recombinant FVO specifically cleaves N-terminal valines and produces hydrogen peroxide. This, in turn, is measured using a horseradish per-oxidase (POD) catalyzed reaction and a suitable chromagen. </div><div class = "text-block"> The HbA1c concentration is expressed directly as %HbA1c by use of a suit-able calibration curve in which the calibrators have values for each level in %HbA1c.</div></div>

[STEPS]
?. Use 250 µl of lysis buffer to lyse 20 µl samples of whole blood and calibrators.IMPORTANT: Make sure the samples are totally lysed. Any solid material floating around will interfere with reading in the platereader.
?. Mix R1a and R1b reagents together in a 70:30 ratio.
?. Add 25 µl of each calibrator and sample to each well.
?. Add 160 µl of reagent R1ab mix to each well. Incubate at 37°C for 5 minutes then read at 720 nm.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 70 µl of R2 to each well. Incubate at 37°C for 3 minutes then read at 720 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: Nested Gibson Assembly
AUTHORS: Erin Garza, Vincent Bielinski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method can be used to increase the efficiency of Gibson Assemblies containing many pieces and/or difficult to assemble DNA fragments.</div></div>

[STEPS]
?. [Select primers]
Using the same primers that you used to amplify your DNA pieces for the Gibson Assembly, you can pick new primer pairs to amplify multiple fragments that have already been ligated together from the failed Gibson reaction.Example: By using the A-Fwd and B-Rev primers in a PCR reaction, fragments A and B can be amplified as one piece.  The C-Fwd and E-Rev primers can also be used to amplify fragments C, D, and E as a single piece.  The Gibson Assembly can then be repeated with a total of 3 fragments instead of 6.  Fragment- 1. A-B                   2. C-E                    3. F
?. [Run a gel]
Run the PCR fragments amplified from step 2 on a .8% - 1% agarose gel to ensure that each of the reactions was successful. Note: If a PCR reaction of some of the pieces failed, then repeat step 1 with different primer pairs to amplify a different set of fragments together.  This also acts as a diagnostic tool to determine which DNA pieces are not assembling efficiently.
?. [Purify DNA ]
Purify the remainder of each of the PCR reactions. We use a Zymo Research DNA Clean and Concentrator kit.
?. [PCR]
Run the PCR reactions using the chosen primer pairs from step 1 and the failed Gibson Assembly as the template.Reaction Mixture25 μl PrimeSTAR Max DNA mastermix22 μl DI water  1 μl Forward primer [10 μM]  1 μl Reverse primer [10 μM]  1 μl Failed Gibson Reaction------------------------------------------------50 μl TotalCycling Conditions98°C for 10 sec.                  Tm-5°C for 15 sec.                   72°C for 10 sec./kb * Run steps 1-3 for 30 cycles
?. [Gibson Assembly]
Set up Gibson Assembly in a PCR tube1 μl each DNA fragment (we dilute each of our fragments to 30 fmol)5 μl Gibson Assembly Master mix (NEB)x μl DI water--------------------10 μl total*Incubate the Gibson Assembly in a thermocycler at 50°C for 1 h.
?. [Transform]
Mix 5 μl Gibson Assembly with 25 μl NEB 5-alpha chemically competent cells and hold on ice 5-10 min
?. [Transform]
Heat shock cells at 45°C for 30 sec and immediately place back on ice for 5 min
?. [Transform]
Add 270 μl SOC to the cells and incubate at 37°C for 1 h
?. [Transform]
Plate 50 μl cells on an LB plate containing the appropriate antibiotics and grow overnight at 37°C
?. [Verify Transformants]
Grow resulting colonies in LB broth + antibiotics and perform colony PCR, restriction digestions, and/or sequencing to verify that the assembled plasmids are correct.
TITLE: Preparing whole cell samples for immunoblotting
AUTHORS: Louise Uoselis, Louise Uoselis

[DESCRIPTION]
Protocol for preparation of HeLa cell lysates for immunoblotting

[STEPS]
1. Add an appropriate volume of 1x LDS Sample Buffer to each sample.
2. Boil each sample at 99 °C with shaking at maximum speed for 10 min 

3. Allow all samples to cool to Room temperature, and quickly centrifuge the samples to collect all liquid in the bottom of the tube. Vortex each sample for ~3 seconds to ensure homogeneity. 

4. Measure the concentration of each sample spectroscopically, blanking with the 1x LDS sample buffer and using an A280 measurement. Dilute samples with 1x LDS Sample buffer to a concentration of < 7 mg/mL if required. 
5. Aliquot out the desired amount of each sample into a separate tube, and add 1x LDS Sample Buffer to each sample to make all samples in the same gel a standard volume. 
6. Either freeze samples at -20 °C until required, or load directly onto a SDS-PAGE gel.
TITLE: Expression and purification of mCherry-NDP52 
AUTHORS: Daniel Bernklau, Dorotea  Fracchiolla, Justyna Sawa-Makarska

[DESCRIPTION]
This protocol describes how to express in E.Coli human NDP52 N-terminally tagged with mCherry and purify it through His affinity purification followed by Size Exclusion Chromatography. 

[STEPS]
SECTION: Expression
1. pETDuet-1_6xHis-TEV-mCh-NDP52 (Addgene  ID: 187829) was transformed into E. coli Rosetta pLySS cells.
SECTION: Expression
2. To express the protein grow E. coli Rosetta pLySS  cells in 4 L of LB medium (w Amp/Cam) at 37°C until an OD600 nm of 0.4. Next, bring the temperature down to 18°C and grow further to an OD600 nm of 0.8. Induce protein expression with 0.5 mM IPTG and grow for further 16 h at 18°C.
SECTION: Expression
3. Pellet the cells at 4000 rpm 4 °C 15 min . Re-suspended the cell pellet in a buffer containing 50mM HEPES pH7.5, 300 mM NaCl, 1 mM MgCl2, 10 mM Imidazole, 2 mM Beta-Mercaptoethanol, cOmplete protease inhibitors (Roche), and DNase (Sigma). Snap freeze in liquid nitrogen and store in -80 °C  until the day of purification.
SECTION: Purification
4. Open the cells by thawing in RT water bath and sonicating 3 x 30 seconds at 50% power using a Bandelin sonicator. 
SECTION: Purification
5. Clear the lysate by ultracentrifugation (40,000 rpm for 45 min at 4°C in a Ti45 rotor, Beckman).
SECTION: Purification
7. Apply to a 5-ml His-Trap column (Cytiva) and elute with a stepwise imidazole gradient (50, 75, 100, 150, 200, and 300 mM). Fractions at 75–100 mM imidazole should contain His-TEV-mCh-NDP52. Pool those fractions and subject to TEV protease cleavage over night at 4°C by very gentle rolling.
SECTION: Purification
6. Filter the supernatant with a 0.45 μm syringe filter on ice.
SECTION: Purification
8. After TEV cleavage, concentrate the protein using a 50kDa cut-off Amicon filter to 0.5 ml and inject onto a Superdex 200 increase 16/600 column (Cytiva) pre-equillibrated with a buffer containing 25mM HEPES pH7.5, 150mM NaCl, 1mM DTT at 4°C.
SECTION: Purification
9. Pool fractions containing pure protein, concentrate using a 50kDa cut-off Amicon filter, snap freeze in liquid nitrogen, and store at −80°C.
TITLE: Surface Density Calculation
AUTHORS: Liv  Jensen

[DESCRIPTION]
This protocol details Surface Density Calculation.

[STEPS]
SECTION: Surface Density Calculation
1. Quantify the fluorescence of a serial dilution of membrane fluorophore and of proteintag fluorophore in 1% SDS-containing buffer (10 micromolar (µM), 6 micromolar (µM), 2 micromolar (µM), 1 micromolar (µM), 0.5 micromolar (µM) of each fluor).

SECTION: Surface Density Calculation
2. Compare the slopes of a plot of fluorescence intensity vs. concentration for the membrane dye and the protein dye to account for differences in optical properties.
SECTION: Surface Density Calculation
3. Quantify the fluorescence of a series of GUV preparations containing varying mole percentage of membrane fluorophore (0.01%, 0.05%, 0.1%, 0.5%, 1%), using a rectangular selection of an approximately horizontal segment of each GUV.
SECTION: Surface Density Calculation
4. Calculate the surface density of membrane dye from known surface area of lipids (~0.7nm2 /lipid).
SECTION: Surface Density Calculation
5. Calculate the slope of a plot of membrane dye surface density vs. fluorescence signal, accounting for intensity from both lipid bilayer leaflets:

  

SECTION: Surface Density Calculation
6. Relate surface density of lipid to surface density of protein label by normalizing to signal from bulk solution (from step 2):
TITLE: Bayesian detection of piecewise linear trends in replicated time-series with application to growth data modelling
AUTHORS: Norman van Rhijn, Panagiotis Papastamoulis, Takanori Furukawa, Magnus Rattray, Mike Bromley, Elaine Bignell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Generally, growth assays for filamentous fungi have been performed on solid media, either as dilution series or spot tests. However, the solid media environment does not accurately mimic the environment encountered during infection (ie the mammalian lung). Previously, we have developed a methodology to perform liquid growth assays in time for </span><span style = "font-style:italic;">A. fumigatus </span><span>and other filamentous fungi including analysis via mathematical modelling.</span></div><div class = "text-block"><span>This protocol is designed for </span><span style = "font-style:italic;">Aspergillus </span><span>species (and other filamentous fungi) to generate growth curves in liquid media in a 96-well plate. This can be done in high-throughput to generate 96 growth curves per run.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines our bacteriophage/virus taxonomy analyses. This analysis includes profiles for the average order relative abundance for each site, the phage species relative abundance profiles for each patient, and the relative abundance of specific species between sites. Intermediate files are also included with the publication, and the paths are specified below. Based on the methods from 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.<br /><br /><br />

[BEFORE_START]
Supplemental 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]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a protocol for high yield recovery of pure DNA from agarose gels, using GeneJET Gel Extraction and DNA Cleanup Micro Kit

[BEFORE_START]
Add 35 ml 100% ethanol to the <strong>Wash Buffer</strong> concentrate. Add 2.5 ml 100% ethanol to the <strong>Prewash Buffer</strong> concentrate.

[GUIDELINES]
The GeneJET Gel Extraction and DNA Cleanup Micro Kit is based on the ability of DNA to bindÂ to silica membranes in the presence of chaotropic salts. DNA adsorbs to the silica membraneÂ while contaminants pass through the column. Alternatively, after electrophoresis to separateÂ the DNA fragments, the band(s) of interest is excised from an agarose gel and dissolved inÂ Extraction Buffer, then mixed with ethanol and loaded on DNA Purification Micro Column.Â Impurities are subsequently removed from the silica membrane by the addition of the PrewashÂ Buffer and Wash Buffer, and the pure DNA is effectively eluted with Elution Buffer. The purifiedÂ DNA is used for a wide variety of downstream applications.

[STEPS]
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TITLE: Imaging and stimulating enteric neurons in the murine large intestine
AUTHORS: Dante  Heredia, Thomas  Gould, Terence K Smith

[DESCRIPTION]
Protocol for imaging neurons within the murine myenteric plexus. 

[GUIDELINES]
This protocol applies to transgenic animals expressing fluorescent calcium indicators in neuronal cells. 

[STEPS]
1. A ventral midline incision is made and the whole colon is carefully excised into a Sylguard lined dissection dish containing oxygenated Krebs-ringer solution.
2. Using scissors, cut along the mesenteric border until the colonic tube is now a flat sheet. 
3. Carefully pin region of interest (proximal, middle or distal) mucosal side down. Allow for end sections of the proximal and distal regions to be pinned mucosal side up to allow for mucosal brush stimulation. Place two platinum electrode wires across serosal layer in perpendicular direction. 
5. Functional imaging was performed on a Nikon Eclipse FN1 upright fluorescence microscope using Nikon Plan Fluor 20x lens. Image sequences were captured using an Photometrics Prime 95B sCMOS camera and captured on a Windows-based PC using Nikon NIS Elements 4.1. Image sequences were recorded at 25 frames per second.
4. Perfuse with oxygenated Krebs-ringers solution at 36-37 degrees Celcius
6. A camel hair paintbrush was used for mucosal stimulation, where each stimulation consisted of a sequence of five brush strokes in approximately 5 seconds. Electronic field stimulation was applied at 20 hertz for 10 seconds; 6 volts .01 millisecond pulse duration.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The monthly quality control is a more extensive quality check of our 3T MRI system but also the other computers in the operator room. </p>
<p> </p>
<p>The monthly quality control consists of the following components:</p>
<ul>
<li>Virus scan on scan-computer</li>
<li>Moving eyetracker-files from DOS to Windows desktop</li>
<li>Calibration of scanner (Philips protocol)</li>
<li>32 channel SNR test</li>
</ul>

[STEPS]
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TITLE: Peritoneal dialysis in extremely and low birth weight infants: a pooled analysis of case reports
AUTHORS: Ioannis Bellos, Vasilios Karageorgiou

[STEPS]
?. Search strategy: Through a structured algorithm search, major databases will be searched from inception to 01 August 2020. The databases will be MEDLINE, Scopus, Web of science, clinicaltrials.gov and Google Scholar. Keywords provided, with ad hoc modification, will include: peritoneal dialysis, neonate, acute kidney injury, renal failure. After screening the titles and abstracts of all retrieved studies, only those deemed relevant will be accessed in full. Those fulfilling the eligibility criteria will be included in the study.
?. Eligibility criteria: Studies including neonates and infants aged
?. Outcomes and Effect Measures: Primary outcomes: Survival (defined as time to death from commencement of PD) and time to renal recovery will be estimated. For both these outcomes, hazard ratios (HR) for 1. EBW vs. LBW, 2. Sepsis vs. other groups will be reported. Secondary outcomes: The risk of 1) obstruction, 2) leakage at any point during PD will be estimated in patients with ELBW and VLBW. For these outcomes, risk ratios (RR) will be reported.
?. Data Analysis: Primary outcomes: Multivariable Cox regression was used for the primary outcomes []. Multivariable extension allows for correcting for confounders. In our case, lower gestational age and current weight have been associated both with birth weight and with decreased survival. Thus, they fulfill the criteria for confounding and were used as covariates in Cox models. Analyses were performed with the R package survival [https://cran.r-project.org/web/packages/survival/survival.pdf]. Secondary outcomes: Logistic regression was used, with obstruction and leakage treated as positive outcomes. Where there was no mention of such a complication, it was assumed that it did not occur. If co-occurring complications were reported, they were analyzed themselves as separate outcomes if data were adequate.
?. Subgroup Analyses: The analyses for the primary and secondary outcomes will be performed in subgroups stratifying by a) type of catheter used, b) a reported diagnosis of RDS.
?. Sensitivity Analyses: As it is expected that patients under PD treatment of longer duration are likely to be expressing patients with a disproportionately worse baseline severity of illness, it is anticipated that PD duration will not be equally allocated between participants with different severities. This phenomenon, referred to as confounding by indication, will be accounted for with propensity score matching (PSM). A further concern is the non-randomized design in all studies, implying residual confounding in other domains. The PSM approach also provides more reliable estimates in such settings. A logistic regression model will be fitted to calculate the predicted probability of a patient being allocated to compared groups, in our case PD duration less or more than 5 hours. Covariates upon which balance is sought will be age, birth weight and etiology (sepsis/other). The output will be a matched in which primary and secondary outcome analyses will be repeated.
TITLE: Prepare agarose pad for microscopy (2022 iGEM)
AUTHORS: Team Fudan iGEM

[DESCRIPTION]
This protocol describe the steps to perform fluorescence microscopy for E. coli

[BEFORE_START]
Culture cells in liquid media

[GUIDELINES]
Cells should be cultured to a late log phase to increase cell number for microscopy.

[STEPS]
SECTION: Cell growth
1. Grow cells to late-log phase density (e.g. OD600 > 0.6).
SECTION: Agarose pads
2. Prepare a 1% agarose in distilled water, by adding sufficient low-melting agarose to water and dissolve agarose by heating in a microwave for 30-60 seconds.
a

SECTION: Microscopy GFP
7. Place the slide onto the microscope stage, with the coverslip facing the objective.
Use a laser at 488 nm to excite GFP.
Record images.
SECTION: Microscopy GFP
8. Analyze the images. For our microscope with 150x objective, the pixel size is 0.0806 µm.
SECTION: Agarose pads
3. Prepare a glass slide with a double adjacent slides barrier (see below), forming a wall around the pad slide. Add slides on top of the poured agarose solution (1-1.2 mL) to allow formation of a smooth surface. It takes time for 1% agarose solution to cool down to ~40 degree, and the solution is viscous but not runny.
SECTION: Agarose pads
4. Wait for 45-60 minutes. Carefully remove adjacent and top slides, leaving the bottom slide.
SECTION: Agarose pads
5. Cut the pad into 15x15 mm square pierces, and carefully transfer the pad pierces to other cleared slide (left one agarose pad on the existing glass slide, if it was clean).
SECTION: Agarose pads
6. Add 2-5 µL bacteria culture onto the agarose pad, and cover it was a 18x18 mm #1.5 square coverslip. Seal with wax if needed.