eamag/protocols-with-steps huggingface dataset:
TITLE: Passaging cancer organoid cultures
AUTHORS: Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the passaging of organoid cultures. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. The team has extensive experience passaging and expanding organoid models. The method described has mainly been used for the passaging cancer organoids with successful propagation of organoids derived from colon, pancreas and oesophageal tumours. </div></div>

[STEPS]
?. [Process Diagram]
?. [Protocol]
Using a P1000 pipette, detach BME2 drops from the plate. (Optional - detach BME2 drops using a cell-scraper; recommended for passaging a large number of plates).
?. [Protocol]
Pipette suspension up and down multiple times to dissociate organoids from BME2 and transfer to an approproate size tube using a P1000 pipette orstripette.
10 mL
?. [Protocol]
Wash wells with TrypLE (approxper well of a 6 well plate) to collect any remaining organoids and add to the collection tube.
1 mL
?. [Protocol]
Incubate at
37 °C
?. [Protocol]
Check organoid suspension under microscope after 5 minutes and then as required to assess the dissociation of organoids. Use a P1000 to pipette the cell suspension up and down to help dissociate organoids. Stop incubation when organoids broken down to small clumps of cells.
Avoid breaking organoids down to single cells
?. [Protocol]
Centrifuge atfor 2 minutes.
Centrifuge: 800 34
?. [Protocol]
Aspirate supernatant to leave organoid cell pellet.
If the organoid suspension does not pellet during centrifugation or there is a grey haze above the pellet (residual BME2), aspirate as much supernatant as possible. Re-suspend in ice cold PBS and repeat centrifugation.
?. [Protocol]
Re-suspend pellet in appropriate amount of 80% BME2 (per well of a 6 well plate).
200 µl
Start by re-suspending pellet in a small volume to break down, then add additional BME2 to required volume.
BME2 must be dispensed as quickly as possible as it will begin to set at room temperature. A cool block could be used to help keep the temperature down while plating.
Avoid creating bubbles while re-suspending pellet.
?. [Protocol]
Using a P200 pipette dispense a total oforganoid/BME2 suspension per well of a 6 well plate as-droplets.
200 µl
10 µl
15 µl
?. [Protocol]
Place in aincubator (5% CO2) for 15-30 minutes to allow BME2 to set.
37 °C
?. [Protocol]
Addof appropriate organoid media per well of a 6 well plate.
2 mL
?. [Protocol]
Return to incubator. Media change twice a week until ready to passage.
?. [Protocol]
Aspirate media from wells and addorTrypLE to each well.
1 mL
2 mL
If any of the following are true collect organoids and media in a tube and centrifuge to pellet before adding TrypLEBME2 droplets detached from plate and floating in mediaLots of cells or organoids floating in the mediaLots of fibroblasts attached to the plate
TITLE: Discharge of LiPo Batteries Using Saltwater Electrolysis
AUTHORS: marshall.bennett

[DESCRIPTION]
Procedure developed using information gathered from the following sources: 
How To Dispose Of Lipo Batteries? - Standard Battery (standardbatteryinc.com)

Discharging & Disposing of LiPo Batteries – HeliDirect Support Center (zendesk.com)

https://www.researchgate.net/profile/Alireza-Bazargan/publication/352042531_Discharge_of_lithium-ion_batteries_in_salt_solutions_for_safer_storage_transport_and_resource_recovery/links/61ebe8f78d338833e3895d56/Discharge-of-lithium-ion-batteries-in-salt-solutions-for-safer-storage-transport-and-resource-recovery.pdf

[STEPS]
2. Dissolve non-iodized salt into water in a bucket following the measurements outlined in the chart below. 

 

1. Place the battery into a container that is sealable with a vented lid, such as a five gallon bucket. 
3. Carefully pour the salt/water solution into the container holding the battery so that it is completely submerged and covered in several inches of water. 
4. Check for activity from the battery and cover the bucket with the vented lid. Bubbles should emerge from the battery's terminal port as electrolysis begins. 
5. Monitor the battery and the lid regularly. It is possible that residue may develop along the surface of the salt/water solution, and it may take several changes of the water for the battery to completely discharge. Complete discharge of the battery could take many days, depending on the size and build of the battery. 
6. Battery is safe to be recycled or disposed of when electrolysis is complete and voltage is at a low level. Store the battery in a sealable plastic bag until it is ready to be disposed of.
TITLE: Laboratory protocols for the detection and genotyping of Cervical high-risk human papillomavirus (HR-HPV) among adult women living in N'Djamena the capital city of Chad.
AUTHORS: Ralph-Sydney Mboumba Bouassa, Zita Aleyo Nodjikouambaye, Damtheou Sadjoli, Chatté Adawaye, Hélène Péré, David Veyer, Mathieu Matta, Leman Robin, Serge Tonen-Wolyec, Ali Mahamat Moussa, Donato Koyalta and Laurent Belec., Laurent Bélec

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Adult Chadian women from 5 of the 10 districts of N’Djamena, the capital city of Chad, were randomly selected for inclusion. Peer educators contacted adult women in community-churches or women association networks to participate to the survey and come to the clinic for women’s sexual health “La Renaissance Plus”, N'Djamena. Medical, socio-demographical and behavioral informations were collected. HPV DNA was detected and genotyped in endocervical swab using Anyplex™ II HPV28 genotyping test (Seegene, Seoul, South Korea).</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><u>Background</u>: Community nasal meningococcal carriage rates are high across Africa.</p>
<p>Meningococcal infections are major causes of morbidity and mortality in the continent;</p>
<p>especially among children and adolescents. This study aimed to determine the prevalence of</p>
<p>nasal carriage and antibiotic susceptibilities of meningococcal isolates from healthy Ethiopian</p>
<p>children and adolescents.</p>
<p><u> </u></p>
<p><u>Method</u>: A cross-sectional study was conducted in one of the sub-cities of Addis</p>
<p>Ababa, Ethiopia. Nasal swabs were collected and processed for identification, serogrouping and</p>
<p>testingsusceptibilities for three antibiotics using standard microbiological techniques. Data on</p>
<p>epidemiologic risk factors were collected using a structured questionnaire and the magnitude of</p>
<p>their association with carriage was assessed using bivariate and multivariate analysis.</p>
<p><u> </u></p>
<p><u>Result</u>: A total of 240 samples were collected (115 from males and 125 from females). The</p>
<p>mean age of study participants was 11.1 years. The prevalence of nasal carriage for <em>Neisseria </em></p>
<p><em>meningitidis</em> was 20.4% (49/240). Carriage was significantly higher among children living under</p>
<p>crowded conditions (OR1.268; 95% CI: 1.186 – 1.355; p = 0.006). The predominant serogroups</p>
<p>were W135 – 20/49 isolates (40.8%) and C - 12/49 isolates (24.5%) and 83.7% of meningococci</p>
<p>were sensitive for Ciprofloxacin. In contrast, isolates showed high resistance to Ceftriaxone</p>
<p>(69.4%) while only 4.2% were sensitive for Penicillin. Multi-drug resistance was documented for</p>
<p>14.3% of the isolates.</p>
<p><u> </u></p>
<p><u>Conclusions</u>: Meningococcal carriage rate was found to be high with higher rates associated</p>
<p>with children and adolescents living in crowded living conditions. Predominant isolates were of</p>
<p>serogroup W135 and C and the isolates showed marked susceptibility to Ciprofloxacin and</p>
<p>resistance to Ceftriaxone and Penicillin.</p>
<p><u> </u></p>
<p><u>Keywords</u>: <em>N. meningitidis</em>, Nasal carriage, Antibiotic susceptibility, Serogroup, Children,</p>
<p>Adolescents, Ethiopia</p>

[STEPS]
TITLE: Enriching and isolating phages in liquid culture
AUTHORS: Adair Borges

[DESCRIPTION]
This protocol explains how we isolate phage from microbial communities using liquid enrichment. For a protocol on enriching and isolating on solid agar plates, check out our companion protocol.

[STEPS]
SECTION: Liquid growth enrichment
1. Set up cultures of your target host strains. We use this protocol for cheese bacterial isolates, which grow best in LB at 25 °C with shaking at 200 rpm, and reach high density after two days. 
SECTION: Liquid growth enrichment
2. After cells have grown to high density, prepare your infection media. We used LB supplemented with 1 mM MgSO4 and 1 mM CaCl2 (final concentrations). Add your phage extracts to this media, either individually for many parallel enrichments, or pooled together.
 
 

SECTION: Liquid growth enrichment
3. Aliquot your infection media into 3 mL glass culture tubes, or deep-well 96-well plates. We generally use 0.25–1 mL of media for these enrichments. 
SECTION: Liquid growth enrichment
4. Subculture your hosts into the infection media at a 1:100 dilution. For instance, you would add 10 µL of host culture to 1 mL of infection media.
SECTION: Liquid growth enrichment
5. Incubate your enrichments at 25 °C with shaking at 200 rpm overnight. 
SECTION: Liquid growth enrichment
6. Harvest infected subcultures: Move infected cultures into 1.5 mL tube + 50 µL CHCl3. Spin down for 5 min at max speed, then transfer the supernatant to fresh 1.5 mL tube + 50 µL CHCl3.
SECTION: Liquid growth enrichment
7. Use uninfected parent cultures to pour phage plates using the double agar overlay method. Add 200 µL of culture to 3 mL of molten (but not overly hot) top agar. Pour top agar onto a room-temperature LB plate, swirling to evenly distribute the agar across the plate before it cools. 

 

SECTION: Liquid growth enrichment
8. Once the plate is cooled, spot the phage-enriched supernatant onto the phage plate. If you only have one enrichment per host, you can use a larger volume (like 10 µL) in one large drop that you can gently swirl with the pipette tip to distribute. If you have many enrichments per host, you should pipette smaller, 2–3 µL spots onto the host in a grid pattern. 
SECTION: Liquid growth enrichment
9. Incubate plates at room temperature. Plaques will show up in 24–72 hours. 
SECTION: Liquid growth enrichment
10. Pick plaques using a pipette tip, and transfer into 300 µL of SM buffer with 50 µL of CHCL3.
SECTION: Liquid growth enrichment
11. Purify these phages by diluting the picked plaque down to a concentrate that will give single plaques, then infecting 200 µL of host culture with 10 µL of diluted phage, and finally plating out using the double agar overlay method. Repeat 2×.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol gives two methods for prophage induction in heterotrophic bacterioplankton; one with no viral reduction and the other with viral reduction (see guidelines).<br /><br />Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30&ndash;33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.

[GUIDELINES]
<strong>Natural populations of heterotrophic bacteria, no viral reduction<br /><br />Materials<br /><br /></strong>-Freshly filtered (0.02 µm; Whatman Anodisc) formaldehyde solution (formalin; 37%);<br />-Mitomycin C (Sigma; 1 mg/mL stock solution, dissolved in deionized water [DI]); <br />-Materials for epifluorescence or flow cytometry enumeration of viral particles; <br />-Electron microscopy–grade glutaraldehyde (Sigma Aldrich).<br /><br /><em>Prophage induction</em>- Procedure in "steps"<br /><br /><strong>Natural populations of heterotrophic bacteria, viral reduction<br /><br />Materials<br /><br /></strong>-Freshly filtered (0.02 µm) formaldehyde; <br />-Mitomycin C (Sigma); <br />-Materials for epifluorescence or flow cytometry enumeration of viral particles; <br />-Cartridge (30- or 100-kDa cutoff) to make virus-free water; <br />-Filtration (0.2 µm pore size) to reduce viral abundance (see also Weinbauer et al., this volume).<br /><br /><em>Prophage induction</em>—The rationale of the virus reduction approach is to avoid new infection by reducing the number of viruses and, thus, the encounter rates with hosts (Weinbauer and Suttle 1996). This can be accomplished by several methods (see Weinbauer et al., this volume). Prokaryotic cells with reduced viral abundance (25–50 mL) are incubated at in situ temperature in triplicates with or without inducing agent C (see also above). Samples for enumeration of prokaryotes and viruses are taken periodically and fixed as described above. Calculation of induced viral production and the percentage of cells containing a prophage (% lysogens) is calculated as described above.<br /><br /><strong>References<br /><br /></strong>Chen, F., J. R. Lu, B. J. Binder, Y. C. Liu, and R. E. Hodson. 2001. Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR Gold. Appl. Environ. Microbiol. 67:539-545.<br /><br />Danovaro, R., and M. Middelboe. 2010. Separation of free virus particles from sediments in aquatic systems, p. 74-81. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. <br /><br />Jiang, S. C., and J. H. Paul. 1996. The abundance of lysogenic bacteria in marine microbial communities as determined by prophage induction. Mar. Ecol. Prog. Ser. 142:27-38.<br /><br />Wommack, K. E., and R. R. Colwell. 2000. Virioplankton: Viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 64:69-114.<strong><br /></strong>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The working concentration of <a href="http://store.p212121.com/blasticidin-s-hcl/">blasticidin S HCl</a> for use in mammalian cell gene selection can range anywhere from 2-100&mu;g/mL depending on the cell lines used.<br /><br />Optimal doses typically range from 2-20&mu;g/mL<br /><br />However, we recommend performing a kill curve to determine the optimal dose for your cell lines.

[STEPS]
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TITLE: High-throughput screening of multiple Caenorhabditis elegans strains
AUTHORS: Tanara Peres

[DESCRIPTION]
I developed this method for growing worms to avoid the steps of bleaching, refeeding and washing worms off plates. When using hundreds of strains these are very time consuming. Eggs will hatch on 24 well plates and worms will be imaged on the same plates. This way one person can easily screen 30 strains per week, with 3 bioreps.

[STEPS]
SECTION: Preparing plates
1. Prepare 60 mm NGM plates for worm maintenance. Dry plates for 1 hour under flow hood. If using 40 strains per week, you need at least 120 seeded plates per week (chunking Monday, Wednesday and Friday).

Always use the same agar, peptone and LB throughout the project (do not change reagents mid project).
SECTION: Maintaining worms
5. Get strains out of freezer and grow them for at least a week without starving. I chunk worms every Monday, Wednesday and Friday. Make a very small chunk on Friday so that they are not starving the following Monday.
SECTION: Imaging on Hydra
7. Allow eggs to hatch and grow on plates for 3 days. Worms should be day 1 adults.
Acclimate worms on imaging room for at least 30 min. Start imaging 24 well plates.
SECTION: Preparing plates
2.  Prepare overnight cultures of OP50 E. coli by picking one colony into a 50 mL Falcon tube containing 30 mL LB Miller. Incubate overnight at 37ºC with 200 rpm shaking.
SECTION: Preparing plates
3. Seed plates with 200 μL liquid culture. Keep plates in cold room until ready to use.
SECTION: Preparing plates
2. 
SECTION: Preparing plates
4. Prepare 24 well plates with peptone free NGM by dispensing 600 μL per well using Integra VIAFILL dispenser. Dry plates for 1 hour under hood. Prepare enough 24 well plates for 2 weeks and keep them in cold room until use. Seed plates with 15 μL OP50 per well the day before experiment.
SECTION: Picking worms
6. Have worms on 60 mm plates with plenty of food and eggs. 24 well plates need to be seeded with 15 μL OP50 the day before and left on bench. Pick 5 to 20 eggs per well. Use 2 rows per strain and save the plate maps to make the metadata. Keep plates at 20ºC for 3 days.

* If there are not enough eggs, pick an adult worm and allow it to lay eggs on wells. Remove the worm.

* Pick eggs Monday, Tuesday and Friday for imaging Thursday, Friday and Monday.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation  of Yeast-Sucrose-Broth, that is used as liquid media for growing <em>Zt.</em> This is called 'YSB' in Bruce McDonald lab.<br /><br />Have a look for example: Zain, M. E., et al. 'Influence of growth medium on diagnostic characters of Aspergillus and Penicillium species.' <em>African Journal of Microbiology Research</em> 3.5 (2009): 280-286.</p>

[STEPS]
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TITLE: Rare Trees Survey outside Plots (Megantic Only)
AUTHORS: Sabine St-Jean

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here, we describe the standardized protocol used by the </span><a href="http://www.caboscience.org" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Canadian Airborne Biodiversity Observatory</span></a><span> (CABO) to survey additional rare trees as needed to ensure that all tree species observed across the Parc national du Mont-Mégantic site are represented by at least 10 Dominant or Co-dominant individuals, in order to fulfill CABO-wide needs for the calibration of sensor data, as mentioned at step 1 of the </span><a href="https://www.protocols.io/view/canopy-trees-survey-protocol-forests-of-southern-q-29bgh2n" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Canopy Trees Survey Protocol - Forests of Southern Québec Protocol</span></a><span>.</span></div></div>

[STEPS]
?. [Tree Selection and Scouting]
Identify the tree species represented by
To do so, export the .csv file from Fulcrum → Vegetation Surveys: Large Trees  → [filter: appropriate site]. Then, in Excel, filter the records by species and Crown class.
?. [Tree Selection and Scouting]
For these species, find individual trees ≥ 9 cm DBH outside the plots to bring the sample size up to at least 10. If possible, use the trees recorded at step 8 of the Canopy Trees Survey Protocol - Forests of Southern Québec Protocol. If not, create Plant records for the selected trees by following the directions from step 8.
?. [Tree Level Measurements]
For these trees, record the required measurements.
?. [Tree Level Measurements]
Save your record.
?. [Tree Level Measurements]
?. [Tree Level Measurements]
Record species identification, DBH, height, and crown dimensions in Plants → Plant Size → Size Measurements. Refer yourself to the Canopy Trees Survey Protocol - Forests of Southern Québec Protocol to know how to take these measurements.By clicking on the Items button, the following screen will show up.Create one new item per measurement.Save your size measurements.
TITLE: Direct RT-qPCR assay for the detection of SARS-CoV-2 in saliva samples
AUTHORS: Francesco Saverio Tarantini, Siyu Wu, Harry Jenkins, Ana Tellechea Lopez, Hannah Tomlin, Ralph Hyde, Katarzyna Lis-Slimak, Jamie Louise Thompson, Sara Pijuan-Galitó, Danielle Scales, Kazuyo Kaneko, Jayasree Dey, Emily Park, Jack Hill, I-Ning Lee, Lara Doolan, Asta Arendt-Tranholm, Chris Denning, Claire Seedhouse, Andrew  V Benest

[DESCRIPTION]
Detection of SARS-COV-2 in Saliva Samples

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient and rapid way to concentrate proteins present in supernatants using Vivaspin columns (3,000 MWCO).</p>

[BEFORE_START]
<p>Prepare a 25 mM Ambic (Ammonium bicarbonate) solution:</p>
<p>19.77 g Ambic in 10 ml Ultrapure H2O.</p>

[STEPS]
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TITLE: Calibrating a pH pmeter
AUTHORS: PMAT0001 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Step-by-step instructions on calibrating a pH meter</div></div>

[STEPS]
?. [Main steps]
Dip in pH 4
?. [Main steps]
Press “Cal”
?. [Main steps]
Press “✔️”
?. [Main steps]
Press “✔️”
?. [Main steps]
Dip in pH 7
?. [Main steps]
Rinse pH meter
?. [Main steps]
Press “✔️”
?. [Main steps]
Dip into sample; Note percentage purity as well
?. [Important notes]
- When liquid level falls way below the "hole" at the top, replenish with some KCl- After finishing experiments for the day, rinse pH meter, secure top with parafilm and put in KCl (parafilm to the KCl storage as well)
TITLE: Quality Indicators for Acute Cardiovascular Diseases: Protocol for Scoping Review
AUTHORS: Koshiro  Kanaoka, Yoshitaka Iwanaga, Yasushi Tsujimoto, Akihiro Shiroshita, Takaaki Suzuki, Michikazu Nakai, Yoshihiro Miyamoto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Abstract</span></div><div class = "text-block"><span style = "font-weight:bold;">Introduction: </span><span>Acute cardiovascular diseases including acute coronary syndrome, acute decompensated heart failure, and acute aortic diseases are leading causes of death in acute clinical settings. Quality indicators are widely used in cardiovascular field to assess and improve the quality of care, but there was no comprehensive review which summarize and assess the currently used quality indicators for acute cardiovascular diseases.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods and analysis: </span><span>The scoping review will be performed according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) statement. The review will include the following five-stage approach by The Joanna Briggs Institute: (1) identifying the research question; (2) identifying relevant studies; (3) study selection; (4) charting the data and (5) collating, summarize and report the results.</span></div><div class = "text-block"><span style = "font-weight:bold;">Ethics and dissemination: </span><span>Since our study is a review of the literature, ethics approval is not indicated. We will present the findings from this study in peer-reviewed journals.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
METAVIR is a web server designed to annotate viral metagenomic sequences (raw reads or assembled contigs). A set of published viromes, identified as "public projects", is already available, and your own data sets can be processed in a private environment.

[GUIDELINES]
<strong>Video tutorial<br /><br /><iframe src="https://www.youtube.com/embed/wdHBe5agYwo" width="560" height="315" frameborder="0" allowfullscreen="allowfullscreen"></iframe><br /></strong>

[STEPS]
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