eamag/protocols-with-steps huggingface dataset:
TITLE: C-19 protocol
AUTHORS: yalhiyari 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A rapid covid screening taking 30sec- 1 min once placed in DOCI machine.  Also does not require any biological sample manipulation.</div></div>

[STEPS]
?. Centrifuge: 3500 33, 5 min, 22 10
?. Need pipet tips and 20-200ul pipet, 96well glass bottom plate Fishersci 07-000-633
[Aliquot 50ul of supernatant 1 well per sample into 96 well glass bottom plate ]
?. Stick 96 well plate into machine for viral detection.  30 sec-1 min per run
TITLE: 3.5 Applications of G-Trap Assay
AUTHORS: Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-</span><span style = "font-style:italic;">S</span><span>-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.</span></div><br/><br/><div class = "text-block"><span>Section 3.5 'Applications of G-Trap Assay' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' </span><a href="https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee</span></a></div></div>

[STEPS]
TITLE: Ferrous Ion-chelating Activity
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The effects of ferrous ion-chelating activity were conducted according to Guo et al.An aliquot of 1 ml extract was added to a solution of 100 µL of FeCl</span><span style = "vertical-align:sub;">3</span><span> (2.0 mM), 3.7 ml of distilled water and 200 µL of ferrozine (5.0 mM). After 20 min, the absorbance was recorded at 562 nm. The inhibition percent and IC</span><span style = "vertical-align:sub;">50</span><span>values were calculated.</span></div></div>

[STEPS]
TITLE: 6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA Neurons
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes 6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA Neurons from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [Cell Seeding]
Plate miDA neurons (3 × 103 per well) in a 384-well plate coated with poly-D-lysine and laminin.
?. [Calcein AM Assay]
Prepare fresh 6-OHDA in vehicle solution (0.15% ascorbic acid in H2O).
Control cells should be treated with vehicle solution alone.
?. [Calcein AM Assay]
Incubate cells with fresh 6-OHDA solution for .
?. [Calcein AM Assay]
Stain live cells with .
[calcein-AM]
The calcein AM assay is based on the conversion of the cell permeant nonfluorescent calcein AM dye to the fluorescent calcein dye by intracellular esterase activity in live cells.
?. [Calcein AM Assay]
Image using a confocal microscope.
?. [Calcein-AM Analysis]
Quantify neurite length using high content analysis software (HCA-Vision V2.2.0. CSIRO).
TITLE: Binding properties of Human IgG to immunoglobulin-binding proteins tested by   double immunodiffusion (Ouchterlony) technique.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">1. IBP-HRP conjugates can be prepared in the laboratory by the periodate method.</div><div class = "text-block">2. The Human IgG used can be commercially available of prepared in the laboratory from human sera by affinity chromatography (using an SpG-agarose column).</div><div class = "text-block">3.  SpG-agarose column are widely commercially prepared.</div><div class = "text-block">4. PURE-1A antibody purification kit will be discontinued. However there are others product for this purpose as Montage Antibody Purification Kit with PROSEP-G media. </div></div>

[STEPS]
?. The binding of Human IgG to peroxidase-conjugated Immunoglobulin-binding protein (IBP-HRP) including SpA-HRP, SpG-HRP, SpL-HRP, SpLA-HRP, SpAG-HRP, SpLG-HRP, SpLAG-HRP and turtle serum (Sigma-Aldrich) as a negative control  are investigated by double immunodiffusion.
?. Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
?. Initially, aliquots of 25 µl of purified human IgG (Sigma-Aldrich) at a concentration of one (1) µg/µl are applied to the centre well. Human IgG could be prepared homemade by purifying human serum by SpA-affinity chromatography (PURE-1A, Sigma-Aldrich).
?. The peripheral wells are filled with 25 µl each of bacterial protein conjugates including SpA-HRP, SpG-HRP, SpL-HRP, SpLA-HRP, SpAG-HRP, SpLG-HRP, SpLAG-HRP diluted 1:5.
?. The gels are incubated at RT for 48−72 hours incubated in a humidity chamber.
?. After that the gels are examined for precipitin lines.
?. Turtle serum is included as negative controls and all IBP-HRP conjugates suppose to react with human IgG.
?. The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
TITLE: Quick Protocol for Monarch® DNA Gel Extraction Kit (NEB #T1020)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the quick version of the Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020). For the full protocol, please click </span><a href="https://www.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span>.</span></div></div>

[STEPS]
?. Excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to a 1.5 ml microfuge tube and weigh the gel slice. Minimize exposure to UV light.
Trim/remove excess agarose and minimize exposure to UV light.
?. Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 μl buffer per 100 μl or 100 mg agarose).
?. Incubate the sample between 37–55°C (typically 50°C), until the gel slice is completely dissolved (generally 5–10 minutes).  The time that takes a gel slice to melt depends on the size of the slice, the temperature used in the incubation as well as the percent agarose used in the gel.  The time recommended above should be used just as a guideline.
For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 μl gel slice: 400 μl Gel Dissolving Buffer: 150 μl water).
?. Insert column into collection tube and load sample onto the column. Spin for 1 minute at 16,000 x g, then discard flow-through.
?. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer (with ethanol added) and spin for 1 minute at 16,000 x g. Discarding flow-through is optional.
?. Repeat Step 5 (Step 5: Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute at 16,000 x g. Discarding flow-through is optional).
?. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
?. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute at 16,000 x g to elute the DNA.
Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>PBCV-1 is a large plaque virus (approximately 2-3 mm in diameter).  The plaque size of the viruses reflects the length of the virus replication cycle.  PBCV-1 has a life cycle of about 6-8 hours.  When titering you should use plates that were poured no more than 1 day before the time of use.  Variable plaque formation will result if fresh plates are not used.  (This is similar to a phage phenomenon.)  Pyrex petri dishes do give more uniform plaque formation versus the plastic, however this is not so critical as with slower growing NC64A viruses.</p>

[GUIDELINES]
<p><strong>MATERIALS:</strong></p>
<p> </p>
<p>         1)      MBBM  plates with MBBM + 1.5% agar</p>
<p>         2)      MBBM soft agar with MBBM + 0.75% agar</p>
<p>         3)      50 mM Tris-HCl, pH 7.8, sterile, for dilution blanks</p>
<p>         4)      13 x 100 mm pyrex tubes, capped and sterile</p>
<p>         5)      50°C water bath</p>
<p>         6)      NC64A culture concentrated to 4.0 X 10<sup>8</sup> cells/ml</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
?. 
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?. 
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?. 
?.
TITLE: Transferring C. elegans to S-basal to grow them in liquid culture
AUTHORS: Muhammad Zaka Asif, Man Shah, Yosef Smadi

[DESCRIPTION]
This protocol describes day 2 of our workflow to grow worms in liquid culture to induce the production of natural products (in this case, 1-HP derivatives). In this protocol, we get an approximate count for the number of life-stage synchronized worms we have, ensure to concentrate them to 30,000 worms/ml, and provide E.coli OP50 for them to be able to progress 

[STEPS]
1. Remove flasks with L1-arrested worms from the shaker
2. Swirl vigorously for at least 30 seconds, then collect a 1 uL aliquot and place onto a glass slide for inspection. Use a 0.5 uL aliquot if there are more than 100 worms in the 1 uL aliquot.
3. Count using a tally counter. Repeat for at least 3 trials (may need to do more if you don’t shake enough initially and end up empty).
4. Once all the aliquots are collected this way, pour each of the flasks volume into a 15 mL flip top tube. Measure the volume. Determine the total population from these two data, as shown below.
5. Total population = (total volume in mL) X (concentration of worms/uL) X 1000 uL/mL
6. Centrifuge the flip top tubes at 1200 rpm for 3 minutes.
7. Aspirate out supernatant, leaving about 0.5 mL behind.
8. Dilute in enough S-basal to achieve a concentration of 30,000 worms/mL.
9. The minimum volume should be 0.5 mL if there are less than 5,000 worms total.
10. Shake vigorously to ensure worms are transferred to solution. Pour into the same flask as before (to minimize transfer loss).
11. Add enough bacteria for a concentration of 2% E. Coli. Make sure the final worm concentration is 30,000 worms/mL (essentially, add less S-basal based on how much bacteria is being added).
12. Repeat steps 1-10 for controls with worms (one for DMSO and one for bacteria only).
13. If using negative controls, repeat steps 9 and 10 (no worms).
14. Essentially, as negative controls, the flasks will have the same total volume of solution (identical S-basal and bacteria) as the earlier flask. They will be exposed to the same conditions just without any worms. The negative controls will later be separated into bacteria-only, bacteria + DMSO, and S-basal + 1-HP in Day 4.
15. Put the flask back in the shaker
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In this protocol,<span lang="en"><span class=""> the extraction of template DNA out of <em>Synechocystis</em> sp. PCC 6803 samples, the PCR of the template and the application on a agarosegel are described. The protocol was handed over by Maximilian Dietsch MSc.<br /></span></span></p>

[BEFORE_START]
<p><strong>Culture</strong> your desired <em>Synechocystis </em>colonies for <strong>at least 7 days</strong> on BG11 agar plates.</p>
<p>You can use the <a href="https://www.protocols.io/view/recipe-for-standard-bg-11-media-gtmbwk6" target="_blank">recipe for standard BG-11 media</a>, which includes the recipe for BG-11 media based agar plates, of Anna Behle MSc.</p>
<p><a href="https://www.protocols.io/view/recipe-for-standard-bg-11-media-gtmbwk6" target="_blank"></a>Always <strong>work under sterile conditions</strong> while inoculating your agar plates!</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Nuclei Isolation from Human Subchondral Bone for 10x Multiome
AUTHORS: Takuya Sakamoto, Merissa Olmer, Martin Lotz

[DESCRIPTION]
This protocol describes isolation of nuclei from fresh-frozen human knee subchondral bone for use in Omics analyses, including RNA-sequencing of ATAC-sequencing. Tissue dissociation was the most critical step in optimization which was monitored by nuclei yield, integrity and purity. Methods for quality control are also described.

[STEPS]
TITLE: Laboratory and radiological data collection
AUTHORS: Kajiru Gad Kilonzo, Stefanie J. Krauth, Jo Halliday, Clive Kelly, Stefan Siebert, Gloria Temu, Christopher Bunn, Nateiya M Yongolo, Sally Wyke, Emma McIntosh, Blandina Mmbaga

[DESCRIPTION]
This protocol details laboratory and radiological data collection.

[STEPS]
SECTION: Laboratory and radiological data collection
1. Following completion of the screening and examinations within both the community and hospital data capture, adult patients with evidence of synovitis or joint swelling will have 20 mL blood taken for later analysis of acute phase reactants (ESR, CRP) and autoantibodies (RF, CCP, antinuclear antibodies, other antibodies as relevant).
SECTION: Laboratory and radiological data collection
2. Patients below 18 years of age will likewise have a maximum of 20 mL of blood taken, but not exceeding 3 mL per kg.

SECTION: Laboratory and radiological data collection
3. Ultrasound will also be performed on all patients with swollen joints and results will be stored securely with the patients’ medical records according to this study’s data management plan.
SECTION: Laboratory and radiological data collection
4. Patients with suspected arthritis will be referred to KCMC Rheumatology clinic for clinical management, which may involve x-ray of hands and other affected joints. 
 

SECTION: Laboratory and radiological data collection
5. All patients with articular abnormalities will also undergo ultrasound examination done by a radiologist, research assistant or physician who have been trained specifically to undertake joint ultrasound examination. 
 

SECTION: Laboratory and radiological data collection
6. Local protocols for clinical decision making informed by these additional diagnostics will be developed by local physicians and paediatricians involved in this study, in consultation with the rheumatologists in Glasgow/Newcastle who are part of the study team, to ensure consistency in clinical process, decision making and sign posting for onwards management.
TITLE: eDNA extraction with kaneka
AUTHORS: Fei  Xia

[DESCRIPTION]
We used the Kaneka Simple DNA Extraction Kit version 2 (Kaneka) for on-site DNA extraction from Sterivex. We injected 500 µl of Solution A of the kit into the Sterivex and shook it by hand for 1 min. We collected the Solution A buffer in the Sterivex by connecting the syringe with the connecting tube. We added 70 µl of Solution B into the collected Solution A buffer in a 1.5-ml microtube and centrifuged (~2,000 g) with a portable centrifuge for 3 min. We collected 200 µl of the supernatant solution.

[STEPS]
1. 

Inject 250 µL of A buffer into the Sterivex, 1 min shake and rotate.

2. 

Put Sterivex into 50ml tube and secured for centrifugation 5000 rpm, 5 min
4. 

Add 40 µL  of B buffer to the 1.5ml tube.

6. 

Store the samples at -20 °C . 

3.  

Remove the liquid from the inlet and put into the 1.5ml tube.
5. 

Mix by vortexing.
TITLE: Keto Blast Gummies Reviews &amp; Shark Tank 2022
AUTHORS: ketoblastiesreviews  

[DESCRIPTION]
Keto Blast Gummies Reviews & Shark Tank 2022


[STEPS]
1. Keto Blast Gummies Reviews & Shark Tank 2022
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Consumption of unhealthy or junk foods leads to multitudinous health problems like cholesterol, BP, diabetes, and heart problems. Piecemeal from other weight loss supplements, one can get several weight loss advantages by using Keto Blast Gummies. Kwazi Keto Gummies These gummies are succulent, natural, and sugar-free for all mortal beings! 
About Keto Blast Gummies 
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Also, this formula may induce lots of energy inside the body to perform physical conditioning. Also, Keto Blast Gummies are perfect for instant weight loss naturally. Because of the herbal factors, this product works naturally without any negative impacts. 
Likewise, it may decline fatigue and frazzle from the body and keep your body energetic throughout the day. Keto Blast Gummies (Kwazi Keto Gummies) may remove fat from all the tubby areas like the belly, shanks, arms, midriff, and others. 
Marvelous Constituents of Keto Blast Gummies 
The seasoning of these gummies are natural and free from any side goods. According to the manufacturer, Keto Blast Gummies contains only herbal constituents like 
BHB (Beta-Hydroxybutyrate) Ketone – Generally, BHB is a kind of ketone that helps to introduce the ketosis process in the body. It's a process that promotes healthy weight loss and allows your body to help adipose cells. 

Official Website:- https://marylandreporter.com/2022/05/20/keto-blast-gummies-reviews-is-kwazi-keto-gummies-helps-in-weight-loss/
TITLE: Naegleria gruberi plasmids
AUTHORS: Anastasios Tsaousis, Eleana Kazana, Tobias von der Haar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Maps and nucleotide sequences of the Naegleria gruberi transfection plasmids (pNaeg-HYG and pNaeg-NEO) reported in Faktorová D. et al. </div></div>

[STEPS]
?.
TITLE: Sample preconditioning before scanning electron microscopy (SEM)
AUTHORS: An.Huang

[DESCRIPTION]
Bacterial samples need to be preconditioned before visualized by scanning electron microscope (SEM). This protocol describes preconditioning methods of bacteria before SEM.

[STEPS]
SECTION: Bacterial samples preconditioning
1. Obtain a small quantity of bacteria to be tested from a culture. 
SECTION: Bacterial samples preconditioning
2. Centrifuge the samples at5000 rpm, 10 min, 4 °C . Resuspend the pellet using 1X phosphate buffed saline (PBS). Repeat this step for three times. Keep the supernatant after the third centrifuge and preserve the samples at 4 °C .

SECTION: SEM sample preconditioning
3. Wash silica wafers with ddH2O and acetone for three times.
SECTION: SEM sample preconditioning
4. Add samples onto the silica wafer using a pipette tip.
SECTION: SEM sample preconditioning
5. Incubate the silica wafers in fixation solution (2.5% glutaraldehyde in 1X PBS) at4 °C overnight.

SECTION: SEM sample preconditioning
6. Incubate the samples in ethanol solution with an increasing concentration (40%, 70%, 96%, 100%) for 60 min each concentration.

SECTION: SEM sample preconditioning
7. Dry the samples at room temperature for 60 min.

SECTION: SEM sample preconditioning
8. Use a precision etching coating system, sputtering the samples with Au-Pd alloy.

 

SECTION: SEM sample preconditioning
9. The samples are ready to be visualized using SEM
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Forms used for data collection in the study entitled: “Risk factors for adverse drug reactions in pediatric inpatients: a cohort study”</p>
<p> </p>
<p>Forms 1 and 2 must be used in the first pharmaceutical consultation after hospital admission of the patient:</p>
<p>i) form 1 is aimed at obtaining information on the prior history of ADR of the patient; and</p>
<p>ii) form 2 is aimed at obtaining information on the prior history of use of the drug.</p>
<p> </p>
<p>Forms 3-6 must be used for the daily recording of information observed in medical prescriptions.</p>
<p>i) form 3 is used to record potential drug interactions;</p>
<p>ii) form 4 for registration of off-label drugs prescribed;</p>
<p>iii) form 5 for the daily recording of the use of drugs; and</p>
<p>iv) form 6 for daily monitoring of prescription errors<strong>.</strong></p>
<p> </p>
<p>Furthermore, form 7 must be used for the registration of abnormal laboratory tests or other suspected adverse events.</p>
<p> </p>

[GUIDELINES]
<p>Forms 1 and 2 must be used in the first pharmaceutical consultation after hospital admission of the patient:</p>
<p>i) form 1 is aimed at obtaining information on the prior history of ADR of the patient; and</p>
<p>ii) form 2 is aimed at obtaining information on the prior history of use of the drug.</p>
<p> </p>
<p>Forms 3-6 must be used for the daily recording of information observed in medical prescriptions.</p>
<p>i) form 3 is used to record potential drug interactions;</p>
<p>ii) form 4 for registration of off-label drugs prescribed;</p>
<p>iii) form 5 for the daily recording of the use of drugs; and</p>
<p>iv) form 6 for daily monitoring of prescription errors<strong>.</strong></p>
<p> </p>
<p>Furthermore, form 7 must be used for the registration of abnormal laboratory tests or other suspected adverse events.</p>
<p> </p>

[STEPS]
TITLE: spyDNA650 staining with CO2 independent media for live endothelium microscopy
AUTHORS: Emir  Bora Akmeriç

[DESCRIPTION]
"spyDNA650 staining with CO2 independent media for live endothelium microscopy"


[STEPS]
SECTION: Staining
1. Dilute 1:1000 SPY650 DNA in CO2 independent media(500 uL total volume for 2 slides, 220 uL per slide and incubate in incubator for 20 mins
SECTION: Staining
2. Gently remove overnight media from the u-slides and add warmed dye-media mix. Check whether monolayer is preserved. incubate for 2-3 hours
SECTION: Flow Unit Equilibration
3. In 7 mL Co2 independent media, dilute 3.5 uL SPY650 dye(1:2000 final concentration)

SECTION: Flow Unit Equilibration
5. Set Ibidi Flow units up for quilibration inside microscope. 
SECTION: Flow Unit Equilibration
7. Run for 1 hour, at 40 mbar and half the cycling time(about 5 secs), pay close attention to whether neither of the syringes run empty and do the clamp test.
SECTION: Flow Unit Equilibration
6. Start the heating of the microscope
SECTION: Flow Unit Equilibration
4. Add 3 mL media the leftwards syringe, 4 mL on the other
SECTION: Microscopy
8. Quickly bring one of the slides to the microscopy room without any washing step and plug it to the flow unit