eamag/protocols-with-steps huggingface dataset:
TITLE: Custom open-chamber microfluidic fabrication
AUTHORS: Quyen  Do, Jimena Baleriola, Nora Bengoa-Vergniory, Richard Wade-Martins

[DESCRIPTION]
This protocol described the fabrication of two- and three-open chambered microfluidics suitable for cell culturing using commercially acquired master mould.

[BEFORE_START]
Always store cut-out devices in ethanol. 

Avoid exposing microfluidic devices, particularly the cell-adherent side, to open air and unnecessary contact. Once sterilised, devices should be handled with care following sterile working techniques.

[STEPS]
SECTION: Casting of microfluidic devices
1. A custom master mould containing both duo- and trio-chambers (Figure 1) is manufactured by and obtained from Microliquid (Spain). 

  
SECTION: Casting of microfluidic devices
2. Mix 46 ml of silicon elastomer SylGARD 184, Dow Corning © with 4 mL of its curing agent (supplied in the same pack), i.e. the 1:10 ratio in a 50 ml falcon. 
SECTION: Casting of microfluidic devices
3. Mix well by inverting the falcon multiple times till a homogenous solution is seen by eye.
SECTION: Casting of microfluidic devices
4. Slowly pour the mixture into the master mould and incubate for 2-2.5 hours at 60ºC.

 

SECTION: Device preparation
5. Manually remove the final cast from the mould using a sharp blade.
SECTION: Device preparation
6. Manually cut out individual devices and immediately wash them with 100% ethanol.
SECTION: Device preparation
7. Air-dry cut-out devices in tissue culture hood.
SECTION: Device preparation
8. Place dry and sterile devices on ethanol – sterilised 19 mm-diameter coverslips to form an instantaneously tight seal. 
SECTION: Device preparation
9. Coat both chambers and microchannel area with Poly-D-Lysin (0.1 mg/ml) overnight followed by Geltrex™ prior to cell culturing.
TITLE: Mitochondrial isolation protocol
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes how to isolate crude mitochondrial fractions from HeLa cells. 

[STEPS]
SECTION: Mitochondrial isolation
1. Seed HeLa cells in 15 cm dishes and grow until confluence. Treat the cells with DMSO or the mitophagy-inducing cocktail Oligomycin/Antimycin A (O/A) if needed.
SECTION: Mitochondrial isolation
2. Collect HeLa cells by trypsinization and resuspension in DMEM medium. Centrifuge the cells at 300 x g, 10 min, 4 °C. 
SECTION: Mitochondrial isolation
3. Remove the PBS and resuspend the cell pellet in 1 mL mitochondrial isolation buffer, which was cooled to 4 °C. 
 

SECTION: Mitochondrial isolation
4. To lyse cells without damaging the mitochondria, pipet the 1 mL cell suspension up and down (15-20x) with a 26.5G needle. 
 


SECTION: Mitochondrial isolation
5. Spin the suspension down at 600 x g, 10 min, 4 °C. 
SECTION: Mitochondrial isolation
5.1. Keep the supernatant and centrifugation 600 x g, 10 min, 4 °C.
 


SECTION: Mitochondrial isolation
6. After two spins at 600 x g, subject the supernatant to 7000 x g, 10 min, 4 °C. 

SECTION: Mitochondrial isolation
6.1. The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1 mL mitochondrial isolation buffer. 
SECTION: Mitochondrial isolation
6.2. Then, centrifugation 7000 x g, 10 min, 4 °C. 
 

SECTION: Mitochondrial isolation
7. After two spins at 7000 x g, subject the resuspended pellet to 10000 x g, 10 min, 4 °C. 
SECTION: Mitochondrial isolation
7.1. The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1 mL mitochondrial isolation buffer. 
SECTION: Mitochondrial isolation
7.2. Then, centrifugation 10000 x g, 10 min, 4 °C. 
SECTION: Mitochondrial isolation
8. After two spins at 10000 x g, the pellet can be resuspended in RIPA lysis buffer or used for further procedures such as mitochondrial import assays, proteinase K protection assays, etc.
SECTION: Mitochondrial isolation
2.1. Resuspend the cell pellet in 1 mL of ice-cold PBS (1x) and spin again for 300 x g, 5 min, 4 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Purpose: To test axenic cultures for purity.  <br />Protocol described in S. Bertillson, O. Berglund, D.M. Karl, S.W. Chisholm (2003).  Elemental composition of marine Prochlorococcus and Synechococcus: Implications for the ecological stoichiometry of the sea.  Limnol Oceanogr 48(5):1721-1731.

[GUIDELINES]
<strong>Formulation:</strong><br />
<table>
<tbody>
<tr>
<td>17 g</td>
<td>BD Difco AC Broth</td>
</tr>
<tr>
<td>20 g</td>
<td>NaCl</td>
</tr>
<tr>
<td colspan="2">Qs to 1 L with distilled H<sub>2</sub>O</td>
</tr>
</tbody>
</table>
<br /><strong><strong>After broth has cooled: <br /></strong></strong>
<table>
<tbody>
<tr>
<td>2 M MgSO<sub>4</sub></td>
<td> (2 M MgSO<sub>4</sub>·7H<sub>2</sub>O = 49.3 g/100 ml H<sub>2</sub>O, dissolve and 0.2 µm filter)</td>
</tr>
<tr>
<td>1 M CaCl<sub>2</sub></td>
<td>(1 M CaCl<sub>2</sub>·2H<sub>2</sub>O = 14.7 g/100 ml H<sub>2</sub>O, dissolve and 0.2 µm filter)</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Cecret Workflow Protocol for SARS-CoV-2 Assembly and Lineage Classification 
AUTHORS: Erin Young, TOAST    CDC Workgroup

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides instructions on how to install the staphb_toolkit and run the Cecret workflow. Cecret produces SARS-CoV-2 consensus sequences from single or paired-end fastq.gz or fastq reads and generates lineage classifications using Pangolin and Nextclade. This document applies to all WGS on the Illumina platform and downstream bioinformatics for CDC partner laboratories.</div></div>

[STEPS]
?. [Load Dependencies]
Load Software Dependencies
The staphb_toolkit requires either singularity or docker, Python 3.6 or greater, and Java version 8 or later. If you do not yet have these installed, do so at this time, and ensure they are in your PATH. Within the CDC high-performance computing cluster, these dependencies can be loaded into the environment using the following GNU module commands.
?. [Install staphb_tookit]
Install the staphb_toolkit which includes the Cecret assembly workflow
?. [Running Cecret Workflow]
Run the Cecret workflow to perform sequence assembly and lineage classification
The input directory for Cecret contains a set of single or paired-end (default) fastq.gz or fastq reads from amplicon prepared libraries. By default, Cecret is configured to use the Arctic SARS-CoV-2 V3 primer set for primer trimming. However, this is customizable if another primer set is used. In this run we set the --profile argument to use Singularity containers but Cecret works with Docker containers as well (--profile docker).
?. [Access Output Files]
Paths for the assembled consensus sequence and the summary report file which contain Pangolin and Nextclade lineage classifications
TITLE: Southern blotting probes for HBV genotype D
AUTHORS: Fumihiko TAKEUCHI

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Southern blotting probes for HBV genotype D.</div><div class = "text-block">HBV cccDNA, rcDNA, and dslDNA can be detected by these probes.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bccja.png" /></div></div>

[STEPS]
?. [PCR reaction]
Prepare the 9 tubes (ccc1-9 probes) and mix the reagent (DO NOT mix other primer set.For example, ccc1 fwd and ccc3 rev.  AB1Ex taq0.5 ul210 X buffer10 ul3dNTPmix8 ul4Template (Supernatant DNA)5 ul5Primer (3 uM)17 ul  X 2 (Forward and Reverse)6DNase free water42.5 ul7Total100 ul
AB1Ex taq0.5 ul210 X buffer10 ul3dNTPmix8 ul4Template (Supernatant DNA)5 ul5Primer (3 uM)17 ul  X 2 (Forward and Reverse)6DNase free water42.5 ul7Total100 ul
?. [PCR reaction]
Amplify cycle 2-4 50 times  AB194 ℃1 min (Hot start)294℃10 sec355℃30 sec472℃24 sec572℃2 min64℃Pause
AB194 ℃1 min (Hot start)294℃10 sec355℃30 sec472℃24 sec572℃2 min64℃Pause
?. [PCR reaction]
PCR product is purified by ethanol purification and gel extraction.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A quick Lowry.Assay for the extraction and determination of total protein from <em>Synechocystis.</em></p>
<p> </p>
<p> </p>
<p><em>Photo credit: Miriam Dreesbach, Institute for Synthetic Microbiology, HHU Düsseldorf</em></p>

[BEFORE_START]
<p>Remeber to prepare BSA standards to absolutely quantify your extracted total protein. A BSA range of 5 µg/ml to 100 µg/ml BSA in water or appropiate media showed good results. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Total Health ACV Keto Gummies (Pros and Cons) Is It Scam Or Trusted?
AUTHORS: andrewfilintof  

[DESCRIPTION]
Total Health ACV Keto Gummies

[STEPS]
1. You could take some time and constancy, to attempt investigate various food sources and diets to find the best weight reduction procedure. Anything weight reduction strategy Total Health ACV Keto Gummies you use, it is important to remain propelled and keep away from common nourishing snares, like eating way an excess of inwardly. Regardless of an eating regimen plan that matches your desires and lifestyle, all plans should stress entire food sources. I'm supportive of any program that advances whole food sources, not exceptionally handled food sources, which's something well known diets can choose. Sustenance methods of reasoning might differ on the food varieties that should be designated, yet this turns out as expected because of the way that the information uncovers that there could be no more excellent method for shedding pounds.
https://www.facebook.com/totalhealthacvketogummies/
https://sites.google.com/view/total-health-acv-keto-gummies-/
https://www.scoop.it/topic/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted
https://techplanet.today/post/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted
https://lexcliq.com/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted/
TITLE: Telerehabilitation in patients with respiratory tract diseases: protocol for a scoping review
AUTHORS: Shunsuke Taito, Kota Yamauchi, Yuki Kataoka

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Background</span></div></div><div class = "text-block">Telerehabilitation is delivery of therapeutic rehabilitation at a distance or offsite using telecommunication technologies. However, telerehabilitation for respiratory diseases and respiratory infections is not known. During the COVID-19 pandemic, telerehabilitation may represent the first option for people at home. The COVID-19 is one of respiratory tract disease, and rapid and widespread transmission was demonstrated. Therefore, it is necessary consider telerehabilitation in patients with respiratory tract disease in the acute phase in hospital as well as chronic phase from the perspective of infection prevention.</div><div class = "text-block"><span style = "font-weight:bold;">Aim</span></div><div class = "text-block">The purpose of this scoping review is to assess the intervention program, safety and feasibility of telerehabilitation for patients with respiratory tract diseases in the COVID-19 era.</div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Methods and analysis</span></div></div><div class = "text-block">We describe this protocol that follows the PRISMA extension for scoping reviews (PRISMA-ScR) statement. We also use the scoping review framework by The Joanna Briggs Institute following five stage approach: (1) identify the research question; (2) identify relevant studies; (3) select studies; (4) chart the data and (5) collating, summarize and report the results.</div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><span style = "font-weight:bold;">Ethics and dissemination</span></div><div class = "text-block">In this scoping review, only data from previously conducted studies will be analyzed. We will publish this scoping review in peer-reviewed journals.</div></div>

[STEPS]
?.
TITLE: YEP Medium
AUTHORS: Roscoff Culture Collection

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Use for </span><span style = "font-style:italic;">Schizochytrium</span></div></div>

[STEPS]
?. Filter 1L of aged seawater (at least two months) on prefilter and 0.2 µm filter
?. Heat seawater during 20min at 100°C
?. To 300 mL of seawater, add :1 g Difco bacto-peptone1 g Bacto yeast extract10 g D-glucose anhydrous
?. Complete final volume to 500mL of seawater
?. Autoclave the medium
?. Under laminar flow hood, filter the medium on 0.2 microns Stericup
TITLE: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
AUTHORS: cgarcia

[DESCRIPTION]
La EIDUCAM planificará y desarrollará la formación transversal anualmente en cursos impartidos de forma intensiva a lo largo de todo el curso para facilitar la planificación temporal de su asistencia por parte de los alumnos. Estas actividades de formación transversal permitirán dar a los doctorandos una visión global, de aquellos aspectos que faciliten su inserción en el mundo profesional.

Entre otros aspectos, se pretende que el alumno fortalezca las competencias y habilidades propuestas tales como comprender las metodologías y técnicas de investigación, los procesos de documentación científica, técnicas y métodos de comunicación científica y redacción de los trabajos de investigación en el idioma habitual del ámbito así como las prácticas del trabajo de investigación responsable. El procedimiento a seguir será el siguiente:

[STEPS]
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
1. Anualmente, durante el mes de diciembre, la EIDUCAM abrirá una convocatoria al profesorado para que realicen propuestas de cursos para el programa de formación transversal del doctorado. Se dará un plazo de un mes.
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
2. Las propuestas deberán contener la siguiente información: nombre del coordinador, programa de curso, sistema de evaluación, créditos, horarios, profesorado y presupuesto. LaEIDUCAM recepcionará las propuestas durante un mes. 
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
3. Laspropuestas recibidas serán evaluadas y aprobadas por la comisión permanente de la EIDUCAM durante el mes de enero.
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
4. A finales del mes de enero la EIDUCAM publicará el calendario de toda la formación ofertada para el curso académico. 
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
5. Los alumnos interesados podrán preinscribirse en la secretaría de la EIDUCAMhasta los 15 días anteriores al inicio del curso.
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
6. El curso se activará 10 días antes de su inicio, en función del número de alumnos y de la rentabilidad económica del mismo. En su caso, la secretaría de la EIDUCAM realizará la siguientes actuaciones:

a. Alta del curso en el Modulo de Formación No reglada de Laurea Academic
b. Difusión de la información del curso propuesto
c. Recepción y mecanización de prescripciones 
d. Matriculación y Generación de recibos de pago
e. Habilitación espacio en campus virtual
f. Expedición credenciales de superación en su caso
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
7. El coordinador será responsable de incluir contenidos y atender a alumnado en el campus virtual, de organizar la impartición y evaluación del curso,y del trasladando del acta de calificaciones oficial a la EIDUCAM. Por otra parte, deberá emitir un certificado de aprovechamiento a cada alumno conforme al modelo establecido.
SECTION: P6- PROCEDIMIENTO DE FORMACIÓN TRANSVERSAL
8. La EIDUCAM tramitará los pagos al profesorado de acuerdo con la normtiva vigente de la UCAM.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: center;"><img style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" width="100" height="90" data-src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" data-ofn="nehirarma-üst-Tr.png" />  </p>
<p style="text-align: center;">Nehir Biyoteknoloji Ltd. <a href="http://www.nehirbt.com.tr/en/" target="_blank">www.nehirbt.com</a></p>

[BEFORE_START]
<p>This protocol is derived from "soft lithography" literature and can be modified by user freely.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient DNA extraction and purification of historical museum hides, which potentially have been chemically tanned. </p>
<p> </p>

[BEFORE_START]
<p>Separate PCR-free facility</p>

[STEPS]
?. 
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TITLE: Artificial colonization of logs with ambrosia beetles
AUTHORS: Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to artificially colonize logs with ambrosia beetles. </div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: SciNote troubleshooting protocol
AUTHORS: Audrey  Apgar

[DESCRIPTION]
This protocol was designed for testing out the SciNote integration with protocols.io - not to be used for actual scientific experiments.

[STEPS]
1. 60 min 
allow vial to thaw
2. 5 mL 
pull out 5mL and dispense into pH cup
3. 
 
calibrate pH meter
4. measure pH sample and record in the results section
TITLE: Oprah Winfrey Keto ACV Gummies: Scam Or Legit Gummy Bears?
AUTHORS: oprahwinfreyscambears  

[DESCRIPTION]
Oprah Winfrey Keto ACV Gummies: Scam Or Legit Gummy Bears?


[STEPS]
1. Oprah Winfrey Keto ACV Gummies: Scam Or Legit Gummy Bears?
Oprah Winfrey Keto Reviews-If you have decided that the time has come to lose a many redundant pounds, know that the path to take is neither easy nor short.
Still, presumably, with the right provocation, If it were enough to suffer exhausting sessions in the spa or confined low-calorie administrations. 
Losing weight, still, is the result of multiple measures, constant commitment, and great provocation. 
Losing redundant pounds means reversing the course; combining a controlled diet with a profitable physical exertion, the energy expenditure exceeds the calories introduced and we begin to lose weight. 
Among the multitudinous salutary administrations in rotation, one of the best known and most effective is really the Keto Diet which is gaining wide acceptance among the public with positive feedback also from assiduity experts. 
Faced with the absence of ingested carbohydrates, the body produces glucose, essential for survival, and begins to affect the reserves of fat to produce energy. In this way, a slimming process begins which affects only the fat pads and not the muscle mass. 
The Keto Diet is extensively used in the field of salutary strategies aimed at reducing weight for substantially aesthetic purposes, but it's also used to combat some pathologies ( similar as some epileptic forms) and forms of severe rotundity. 
Of course, the Keto Diet can be tiring for the body especially during the maximum moment of product of ketogenic bodies; for this reason, it's judicious to combine the chosen diet with a food supplement able of enhancing the salutary goods of the diet and neutralizing the general sense of fatigue. 
So let’s find out what Oprah Winfrey Keto is, its main characteristics, and what its fabulous action is. 
WHAT IS OPRAH WINFREY KETO – FULL REVIEWS 
Veritably analogous to the Oprah Winfrey Keto food supplement, it's a fully natural supplement whose abecedarian purpose is to enhance the salutary goods of the Keto diet and to combat the side goods that can do including lack of energy, somnolence, headache, and gastrointestinal diseases. 

Official Website:- https://freaknewswire.com/index.php/2022/05/13/oprah-winfrey-keto-pills-reviews-price-benefits/
TITLE: Human RBM3 ELISA
AUTHORS: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt

[STEPS]
?. Prepare RBM3 standards in RBM3 Dilution Buffer: 0, 31.25, 62.5, 125, 250, 500, 1000 and 2000 pg/ml.
?. Dilute the serum samples 1:10 with RBM3-SAMPLE-BUF.
?. Pipette 50μl/well RBM3-Standards (0-2000 pg/ml) and 50μl/well diluted samples in duplicates onto 96-well microplate.
?. Pipette 50μl/well detection conjugate – diluted RBM3-HRP conjugate onto 96-well microplate.
?. Shake for 5 min and incubate for 120 min at 20-25 °C.
?. Wash 3 times with 1:10 diluted Wash solution.
?. Pipette 50μl/well Tetramethylbenzidin solution and incubate for 30 min at 20-25 °C in the dark on a shaker.
?. Pipette 50μl/well Stop Reagent.
?. Measure absorbance at 450 nm using a plate reader.
TITLE: Enzymatically Cleavable Linkers
AUTHORS: BOC      Sciences

[DESCRIPTION]
Enzyme-cleavable linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release payloads in the lysosomes of target cells. BOC Sciencesis committed to providing flexible ADC enzymatically cleavable linkers at competitive prices. In BOC Sciences, we support various ADC linkers development and bioconjugation services for global customers to achieve new drug discovery milestones with comprehensive and advanced platforms.

[STEPS]