eamag/protocols-with-steps huggingface dataset:
TITLE: PDI variants expression and purification protocol
AUTHORS: Shihui Guo

[STEPS]
?. Human PDI was clone in the pT7-FLAG-SBP-1vector (sigma), designed for expression in E.coli BL21 (DE3) T1 cells (sigma).
?. Inoculate 10-ml Transformed E.coli BL21 (DE3) T1 cells on the medium containing ampicillin.
?. Grow the culture  O/N at 37degree.
?. Transfer 10ml cell culture to 1Liter LB medium (100μg/ml Amp) and shake at 200rpm, 37 degrees.
?. When OD600 reaches 0.4-0.6, add IPTG to a final concentration of 1mM.
?. Keep the induction temperatures at 22-25 degrees for 13hours.
?. Harvest cells at . And this can be store at -20degrees until needed
Centrifuge: 7000 33, 10 min, 4 10
?. Suspend harvest cells in 30ml buffer containing 1U/ml Nuclease, 50 µg/mL DNase, 200 µg/mL Lysozyme,1mM PMSF, 2mM DTT, 2mM EDTA and 1tablet Protease inhibitor cocktail. Alternatively, B-PER reagent can be used to solubilize and lyse the cells.
?. After 30min incubation, sonicate the mixture 10 sec at intensity 10W, 10 times. This will reduce the viscosity.
?. After centrifugation at 11000rpm for 30min, load the soluble supernatant to High CapacityStreptavidin Agarose column pre-equilibrium with wash buffer consist of 20mMTris HCl pH 8.0, 300mM NaCl, 2mM EDTA, 2mM DTT.
?. The target protein was eluted with 2mM Biotin in the wash buffer followed extensively wash.
?. Dialyze the eluted protein subsequently exhaustively against 1xPBS, pH7.4. It is recommended to have three times dialysis with a minimum of 2-4 hs each.
?. Determine the protein concentration using the mass extinction coefficient of 8.62 at 280 nm.
?. Check the purity of recombinant PDIs by SDS-PAGE using a 7.5% gel.
?. Monitor the enzymatic activities of the PDI variants using the insulin reductase assay.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.</strong></p>
<p> </p>
<p><strong>15 Hourly Timepoints: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14</strong></p>
<p> </p>
<p><em>Sampling is for qPCR assay to determine total extracellular phage (qEXT), and for MPN (most probable number) assay to determine % phage infectivity</em>.</p>
<p> </p>
<p><u>For qEXT and MPN sampling</u>, <strong>250 µL of each sample in duplicates</strong> were collected and filtered through a 0.2 µm, 96-well filter plate, for each time point. The flow through filtrate containing phage was stored at 4°C as the sample to analyze.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: In vitro kinase assay
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes in vitro kinase assay.

[STEPS]
SECTION: In vitro kinase assay
1. Mix recombinant proteins TBK1 or ULK1-complex (composed of ULK1, FIP200, ATG13, and ATG101) and NAP1 in kinase buffer.
SECTION: In vitro kinase assay
2. Use the kinases at 50 nanomolar (nM) and mix with 250 nanomolar (nM) NAP1.

SECTION: In vitro kinase assay
3. Start the kinase reactions by the adding 2x ATP/MgCl2 kinase buffer to a final concentration of 10 millimolar (mM) MgCl2 and 100 millimolar (mM) ATP.

SECTION: In vitro kinase assay
4. Prepare protein mixtures as master mixes and divide over the number of time points. 
SECTION: In vitro kinase assay
5. To control for potential protein instability, induce the latest time point first and then go gradually to the shortest time point.
SECTION: In vitro kinase assay
6. In this way, keep all protein mixtures at Room temperature for the same time, and terminate the reactions together.
SECTION: In vitro kinase assay
7. Achieve the termination of reactions by the addition of 6x Protein Loading dye and heat inactivation at 95 °C for 5 min.

SECTION: In vitro kinase assay
8. Separate the samples on 4-12% SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
SECTION: In vitro kinase assay
9. After the run, either stain the SDS-PAGE gel with Coomassie or transfer to nitrocellulose membranes for western blot analysis. 
SECTION: In vitro kinase assay
10. In the case of Coomassie staining, incubate the gel for 10 min in Coomassie solution, fix for 10 min with fixation solution, and then destain it 10 min in dH2O. 
SECTION: In vitro kinase assay
11. Cut the band corresponding to NAP1 from the gel with a fresh scalpel and submit for mass spectrometry analysis.
SECTION: In vitro kinase assay
12. In the case of western blotting, transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 60 min at 4 °C using the Mini Trans-Blot Cell (Bio-Rad). 
SECTION: In vitro kinase assay
13. Process the membranes further for western blot analysis, as described in the western blot protocol.
TITLE: FCMPASS - Performing and reporting calibration
AUTHORS: Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps required to calculate the calibration parameters and convert fcs files using the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.</div></div>

[STEPS]
?. Upon completing fluorescence and/or light scatter calibration steps click the ‘Calibrate’ button.
?. The FCMPASS software will now perform fluorescence and light scatter calibration. An FCMPASS export folder will be created in the directory from which the fcs files were imported. This folder will contain calibrated fcs files, a MIFlowCyt-EV report with fields relevant to fluorescence and light scatter calibration complete and supplementary sheets for reproducing the calibration. A calibration output report file will also be generated that contains the relevant figures to support the fluorescence calibration and light scatter calibration that was performed. All of these files should be kept together when shared.
?. The remaining fields within the MIFlowCyt-EV report should be completed as recommended in the associated position paper.
TITLE: Mechanical liver dissociation
AUTHORS: Dorien De Pooter, Ben De Clerck, Koen Dockx, Domenica De Santis, Sarah Sauviller, Pascale Dehertogh, Matthias Beyens, Isabelle Bergiers, Isabel Nájera, Ellen Van Gulck, Nádia Conceição-Neto, Wim Pierson

[DESCRIPTION]
This protocol details the mechanical dissociation of liver.


[STEPS]
SECTION: Reagent preparation
1. Add 55 mL of FCS to a bottle of 500 mL of RPMI1640 with L-Glutamine.

SECTION: Reagent preparation
2. Prepare GentleMACS C-tubes by filling them with 9 mL RPMI 1640 medium + 10% FCS and store the tubes at 4 °C.

SECTION: Reagent preparation
3. Fill the syringe with 20 mLPBS and connect a 23G needle to the syringe.

SECTION: Procedure
4. 
 

Euthanize the mice by decapitation.
SECTION: Procedure
5. Position mouse in dorsal recumbency and pin down. Spray the abdominal and thoracic region with 70% EtOH.
SECTION: Procedure
6. Make a parallel incision from the caudal to cranial side starting from the mouse’s abdomen until the thoracic region.
SECTION: Procedure
7. Gently move the intestines to the right side to get access to the liver using forceps and carefully flip the median lobe and left lobe to the cranial side to visualize the portal vein. 
SECTION: Procedure
8. Insert the tip of the needle in the hepatic portal vein. Slowly push 20 mL PBS through.

 


SECTION: Procedure
9. Carefully dissect the perfused liver and transfer into the C-tube containing the medium.

 

SECTION: Procedure
10. Tightly close C-tube and attach it upside down onto the GentleMACS Octo Dissociator.
SECTION: Procedure
11. Run program m_spleen_04 twice.
SECTION: Procedure
12. After termination of the program, detach C-tube from the GentleMACS Octo Dissociator and keep the tubes on ice until further processing.
SECTION: Procedure
13. Dispose the needle in an approved sharps container.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host’s gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum <em>Firmicutes</em> was most abundant in both avian species whereas Phylum <em>Actinobacteria</em> was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity.</p>

[STEPS]
TITLE: Childhood Obesity Policy Document Analysis
AUTHORS: NotreallyItzhak Yanovitzky, Matthew S. Weber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Evidence-based decision-making is critical to the formulation of effective policy and practice, but despite efforts to increase the availability of research-based knowledge in the policymaking domain, use of research evidence by policymakers has been infrequent, inconsistent, and at times misinformed. The primary objective of this study is to test the idea that the news media have an important role in connecting policymakers to policy-relevant research evidence. Specifically, this project explores three research questions:</div><div class = "text-block">1.	What factors influence policymakers’ acquisition, adoption, and use of research evidence in news reports?</div><div class = "text-block">2.	In what ways, direct and indirect, does the inclusion and representation of policy-relevant research evidence in news reports facilitate the use of research evidence by policymakers?</div><div class = "text-block">3.	What, if any, is the relative contribution of policy-relevant research evidence in news media to policymakers’ use of research evidence in policymaking processes?</div><div class = "text-block">To answer these questions, this study will track federal policymakers’ (i.e., legislators in Congress and government bureaucrats) use of research evidence by collecting, coding, and analyzing a comprehensive set of publically available policy documents (e.g., transcripts of congressional bills, committees/sub-committees hearings, testimonials, floor debates, government-issued memos and reports, transcripts of public speeches or comments) on the topic of childhood obesity, focusing on the scope, type, context, and timing of policymakers’ use of research evidence relative to the progression of the legislative process (see Appendix A) for each of four key federal policies from 2000-2014:</div><div class = "text-block">1.	School nutrition;</div><div class = "text-block">2.	School physical education and activity standards;</div><div class = "text-block">3.	Consumption of sugary snacks and drinks; and</div><div class = "text-block">4.	Advertising and marketing food to children.</div></div>

[STEPS]
TITLE: Polymerase Chain Reaction
AUTHORS: Lis Rocha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This was the PCR protocol used for each set of primers in the study Comparative genomics of </span><span style = "font-style:italic;">Staphylococcus aureus </span><span>associated with subclinical and clinical bovine mastitis (Rocha et al., 2019)</span></div></div>

[STEPS]
?. [primers cl3309sub F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3309sub F/R]
Anealing: 55 °C for 45s
?. [primers cl3309sub F/R]
Extension: 72 °C for 45 s
?. [primers cl3309sub F/R]
final extension at 72.0 °C for 10 min
?. [primers cl3316F/R]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3316F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3316F/R]
Annealing: 55 °C for 45 s
?. [primers cl3316F/R]
Extension: 72 °C for 45 s
?. [primers cl3316F/R]
final extension at 72.0 °C for 10 min.
?. [primers cl3700 - LipoP FCS/RC]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3700 - LipoP FCS/RC]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3700 - LipoP FCS/RC]
Annealing: 54 °C for 45 s
?. [primers cl3700 - LipoP FCS/RC]
Extension: 72 °C for 45 s
?. [primers cl3700 - LipoP FCS/RC]
final extension at 72.0 °C for 10 min.
?. [cl33009cli F/R]
initial denaturation: 95.0 °C for 5 min;
?. [cl33009cli F/R]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [cl33009cli F/R]
Annealing: 45 °C for 45 s
?. [cl33009cli F/R]
Extension: 72 °C for 30 s
?. [cl33009cli F/R]
final extension at 72.0 °C for 10 min.
?. [primers cl3700 - LipoP FCS/RCS]
initial denaturation: 95.0 °C for 5 min;
?. [primers cl3700 - LipoP FCS/RCS]
35 cycles of denaturation at 95.0 °C for 45 s,
?. [primers cl3700 - LipoP FCS/RCS]
Annealing: 50 °C for 45 s
?. [primers cl3700 - LipoP FCS/RCS]
Extension: 72 °C for 1min
?. [primers cl3700 - LipoP FCS/RCS]
final extension at 72.0 °C for 10 min.
?. [Analyzing the amplified fragments]
Analyze the amplicons by electrophoresis in 1X Tris-acetate-EDTA on a 1.0% agarose gel  and visualizeimagen under UV light after staining with 2 mg.ml-1 ethidium bromide.
?. [primers cl3309sub F/R]
Initial denaturation: 95.0 °C for 5 min;
TITLE: 10 Years of Science at Tetiaroa
AUTHORS: Erin Robinson

[DESCRIPTION]
This is a protocol documenting the proposed approach to conducting a scientific research review at a field station. 

[STEPS]
SECTION: Document Tetiaroa Projects
1. Identify the projects with Tetiaroa Ecostation leadership, staff, and researchers
SECTION: Document Tetiaroa Projects
2. Review the RAMS applications for each project 
SECTION: Document Tetiaroa Projects
3. Create (or update) a landing page for the project with a DOI, a short description, and connections to the team, their affiliations, funding, keywords, and the field station. These projects will show up in the DataCite Commons page for the Tetiaroa Society: https://commons.datacite.org/ror.org/04p8xrf95
SECTION: Document Tetiaroa Projects
6. 
SECTION: Document Tetiaroa Projects
5. 
SECTION: Document Tetiaroa Projects
4. Begin draft of review paper for the project using the template attached 
SECTION: Document Tetiaroa Projects
4.1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol gives basic instructions and advice to beginners on tipping Drosophila flies between food vials when maintaining stock lines or during an experiment. It will be helpful to read this before commencing your stock care or experiment, however the best way to get better at tipping flies is with practice!</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: XPRIZE SHINE - Paper-based SARS-CoV-2 NP  Test
AUTHORS: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to perform a SHINE paper-based assay to detect SARS-CoV-2 RNA from a self-collected nasopharyngeal sample. This protocol is intended for in-home use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, </span><span style = "font-style:italic;">et al</span><span>. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. </span><span style = "font-style:italic;">bioRxiv</span><span> (2020). </span></div></div>

[STEPS]
?. [Sample Collection and Viral Lysis]
Open the nasopharyngeal (NP) collection tube and rotate the nasal swab (attached to the NP collection tube cap) 4 times around the inside of each nostril. Return the swab to the collection tube and cap the tube.    Nasopharyngeal collection tube contains necessary volume of FastAmp® Viral and Cell Solution.
?. [Sample Collection and Viral Lysis]
Mix NP sample and FastAmp® Viral and Cell Solution by shaking the closed sample collection tube for
?. [Sample Collection and Viral Lysis]
Wait , incubating sample at , before proceeding to Step 4.
0 Room temperature
?. [SARS-CoV-2 Detection]
Add the entire volume in the dropper bottle containing Reagent Mix A to the tube containing lyophilized Reagent Mix B. Cap the tube.
?. [SARS-CoV-2 Detection]
Mix Reagent Mix A and B by shaking for approximately .
?. [SARS-CoV-2 Detection]
Dip the inoculation loop into the sample collection tube (a small layer of liquid should be contained within the loop).
?. [SARS-CoV-2 Detection]
Transfer liquid in inoculation loop to the Reagent Mix A and B tube by dipping the loop into tube and stirring for . Remove and discard the inoculation loop and cap the tube.
?. [SARS-CoV-2 Detection]
Mix the sample combined with Reagent Mix A and B by shaking for approximately .
?. [SARS-CoV-2 Detection]
Wait  , incubating sample at , before proceeding to Step 10.
0 Room temperature
?. [Paper-based Readout and Automated Analysis]
Open the Sample-Reagent Mix A and B tube and place the test strip into the liquid with the arrows on the test strip pointing upward and towards you.
?. [Paper-based Readout and Automated Analysis]
Wait  with sample at  for visible horizontal bands to appear on the test strip.
0 Room temperature
?. [Paper-based Readout and Automated Analysis]
With the user-provided smart device such as a smartphone, open the HandLens application and select paper-based as the test type.
?. [Paper-based Readout and Automated Analysis]
Take a photo of the test strip, and select upload. The result of the test will appear on the smart device screen.
TITLE: Zymoclean Gel DNA Recovery--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Product Description</span></div><div class = "text-block">The Zymoclean™ Gel DNA Recovery Kit provides a hassle-free method for high yield recovery of pure DNA from agarose gels. Simply add the specially formulated Agarose Dissolving Buffer (ADB) to the gel slice containing your DNA sample, let dissolve, and then transfer to the supplied Zymo-Spin™ Column. There is no need for organic denaturants or  chloroform. Instead, the product utilizes Fast-Spin column technology to yield high-quality DNA in just 15 minutes. DNA</div><div class = "text-block">purified using the Zymoclean™ Gel DNA Recovery Kit is perfectly suited for use in DNA ligation reactions, sequencing, DNA labeling reactions, PCR, etc.</div></div>

[STEPS]
?. [Gel purification from agarose gel]
Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube.
The amount of agarose excised from the gel should be as small as possible. Be sure to excise the desired band with very little or no extra agarose gel.
?. [Gel purification from agarose gel]
Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g., for 100 μl (mg) of agarose gel slice add 300 μl of ADB).
?. [Gel purification from agarose gel]
Incubate at 37-55 C for 5-10 minutes until the gel slice is completely dissolved2. For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 μl agarose, 300 μl ADB, and 100 μl water).
Do not incubate above 60C. It is important that the gel slice dissolve completely. This can be facilitated by gentle mixing during the incubation.
?. [Gel purification from agarose gel]
Transfer the melted agarose solution to a Zymo-Spin™ Column in a provided Collection Tube.
?. [Gel purification from agarose gel]
Centrifuge for 30-60 seconds. Discard the flow-through.
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.
?. [Gel purification from agarose gel]
Add 200 μl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. Repeat the wash step.
?. [Gel purification from agarose gel]
Add ≥ 6 μl DNA Elution Buffer or water directly to the column matrix. Place column into a 1.5 ml tube and centrifuge for 30-60 seconds to elute DNA. Ultra-pure DNA is now ready for use.
DNA Elution Buffer: 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA.Elution of DNA from the column is dependent on pH and temperature. If water is used, make sure the pH is >6.0. Waiting 1 minute prior to elution may improve the yield of larger (> 6 kb) DNA. For even larger DNA (> 10 kb), the total yield may be improved by eluting the DNA with 60-70°C DNA Elution Buffer.
TITLE:  Preparation of pharmacological agents 
AUTHORS: Alexandra Nelson, Allison Girasole, Michael Ryan

[DESCRIPTION]
This is a description of how we prepare different drugs for use in live animals (in vivo) or in brain slices (ex vivo).

[STEPS]
SECTION: This protocol provides a description of the preparation, dilution, application and administration of pharmacological agents.
1. 6-OHDA (Sigma Aldrich) for MFB dopamine depletions was prepared at 5 μg/μL in
normal saline solution.
2. Levodopa (Sigma Aldrich) was administered with benserazide (Sigma Aldrich) and prepared
in normal saline solution. Levodopa (5–10 mg/kg) and benserazide (2.5-5 mg/kg) were given via IP
injection 5–7 days per week over the course of the experiment. 
3. 4-hydroxytamoxifen (4-OHT, 50 mg/kg in Chen oil, IP) was prepared as previously
described (Guenthner et al, 2016; Girasole et al, 2018). To prepare a 20 mg/mL stock
in ethanol of 4-OHT, 4-OHT was added to 200 proof ethanol, vortexed, and placed
on a horizontal shaker at 37°C for 30 min or until the 4-OHT dissolved.
The stock solution was kept covered in foil to minimize light exposure. Next,
to prepare a 10 mg/mL working solution in oil, the 4-OHT/ethanol mixture
was combined with Chen Oil (a mixture of 4 parts sunflower
seed oil and 1 part castor oil) and placed into 1.5 mL Eppendorf
tubes. The tubes were vigorously mixed, wrapped in foil, and left on a nutator
for 45 min at room temperature, vortexed and shaken periodically. The
tubes were then placed in a speed-vac for 2-3 h to evaporate the ethanol.
If necessary, the final volume was adjusted with Chen Oil to 1 mL to reach
a final concentration of 10 mg/mL. 
4. Picrotoxin (Sigma) was dissolved in warm water to prepare a 5 mM stock
solution. Picrotoxin stock which was subsequently diluted in ACSF for a final concentration of
50 μM in ex vivo (slice) electrophysiology experiments.
5. Tetrodotoxin (TTX, Abcam) was dissolved in water at a stock concentration of 1 mM and
added to ACSF for a final concentration of 1 μM in ex vivo (slice) electrophysiology experiments.
6. SKF 81297 (Tocris) was dissolved in water at a concentration of 1mM and added to
ACSF for a final concentration of 5 μM in ex vivo (slice) electrophysiology experiments.