eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol: Association between visual acuity and subsequent psychotic-like experiences in the Avon Longitudinal Study of Parents and Children
AUTHORS: Natalie Shoham

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for a planned secondary analysis of longitudinal data, investigating whether there is an association between visual acuity and future psychotic symptoms in children.</div></div>

[STEPS]
TITLE: Streptococcal Protein-G and Protein-LG sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between SpG) and SpLG) with different immunoglobulin preparations from mammalian and avian species. </div></div>

[STEPS]
?. This ELISA was used to study the interactions between SpG) and SpLG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpLG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.36.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
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<p>This protocol was designed and developed at this laboratory and incorporates a previously published oligoprobe (see below).</p>
<p>The protocol specifically aims to amplify DENV02 viruses and not other dengue viruses. The assay targets the capsid region and is designed as a qualitative test for investigating suspected human cases of DENV-2 infections.</p>
<p>This assay has been superseded by the <a href="https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw" target="_blank" rel="noopener noreferrer"><span class="">Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assa</span>y.</a></p>
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[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
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</div>

[GUIDELINES]
<p>This was a past assay that we no longer in use.</p>
<p>For the best DENV-2 TaqMan assay, please refer to our recommended protocol:</p>
<p>Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay</p>
<p><a href="https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw " target="_blank" rel="noopener noreferrer">https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw </a></p>

[STEPS]
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TITLE:  Isolation of Shigella pathogens from oysters
AUTHORS: Sade Aisha Folashade John, Patrick E. Akpaka, Chandrashekhar Unakal, Arvind Kurhade, Angel Justiz-Vaillant

[STEPS]
?. The filtered homogenate was streaked using standardized loops on Salmonella-Shigella (SS) agar, the loops were flamed periodically to ensure sterility. This was done in duplicate. The plates were then incubated at 34°C. After an overnight incubation at 34°C, the plate with the best significant and adequate colonies was used to test for the presence of Shigella. Those colonies that were morphologically characteristic for Shigella were gram stained:
?. Each slide was labelled according to location and given a number. A circle using a wax pencil was drawn on the underside of each slide.
?. Drop of sterile saline solution was placed into the circle and using a sterile loop, a single colony from the plate was taken and mixed into it.
?. The smear was allowed to air dry and then heat fixed by holding the  slide on  one side with a sterile forcep and passing the entire slide through the flame of a Bunsen burner two or three times with the smear side up. Making sure that overheating does not occur.
?. The heat fixed slide was placed on a straining tray.
?. The slide was gently flood with crystal violet and left to stand for 1 minute.
?. Using a wash bottle of distilled water, the slide was tipped slightly and gently rinsed.
?. Then using Gram's iodine, the slide was gently flood and let stand for 1 minute.
?. The slide was tipped slightly and gently rinsed with distilled was using a wash bottle.
?. 95% ethyl alcohol was used to decolorize the smear by rinsing the slide for 5-10 seconds.
?. The slide was then immediately rinsed with water.
?. Using Safranin, the slide was gently flood to counterstain and let stand for 45 seconds.
?. The slide was tipped slightly and gently rinsed with distilled was using a wash bottle.
?. The slide was then blow dried with bibulous paper.
?. Using a light microscope under oil immersion, the smear was observed to the determine whether the colonies were gram negative or gram positive.
?. This was done per presumptive Shigella colony observed on each plate.
?. A single colony of presumptive Shigella was then subcultured on SS agar and incubated at 34°C prior to biochemical testing. These pure single colonies were further analysed using biochemical tests (Indole, Citrate, Urease, Motility and Triple Sugar Iron Agar). The tests were done together with a control Shigella colony taken from the laboratory in order to make a comparison. A colony of the organism was then saved in a cryovial containing Nutrient Agar -70 °C. This was done per Shigella spp. found.
TITLE: In Silico Molecular Modeling of the Sodium Iodide Symporter from Homo sapiens
AUTHORS: Hristina R. Zhekova, Sergei Y. Noskov

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;:UNDERLINE;">In Silico</span><span style = ":UNDERLINE;"> Molecular Modeling of the Sodium Iodide Symporter from </span><span style = "font-style:italic;:UNDERLINE;">Homo sapiens</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Yeast is a well-characterized genome due to its small size and historical significance in genetics.  The website http://yeastgenome.org/ is a dedicated resource for yeast genomics.</p>

[STEPS]
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TITLE: Síndrome dos ovários policísticos: suas implicações nos  distúrbios metabólicos
AUTHORS: Bárbara Karoline Rêgo Beserra Alves, Leandra Caline dos Santos, Antônio Jason Gonçalves da Costa, Maria Rosiany Sousa Moreira, Gleyson Moura dos Santos, Paulo Víctor de Lima e Sousa, Nara Vanessa dos Anjos Barros

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A Síndrome do Ovário Policístico (SOP) compreende uma desordem endócrino-metabólica na idade reprodutiva, com incidência de 6 a 10% das mulheres no período fértil. As diversas manifestações clínicas desta doença associam-se com distúrbios metabólicos como à resistência insulínica (RI), aumentando significativamente o risco de doenças cardiovasculares (DCV), diabetes mellitus tipo 2 (DM 2) e dislipidemias. O objetivo do estudo foi revisar o perfil metabólico em portadoras de SOP, as consequências e estratégias nutricionais para prevenção, tratamento e controle dessas desordens metabólicas. Realizou-se uma revisão bibliográfica, com um total de 22 artigos científicos, publicados entre os anos de 2004 a 2015, nas bases de dados Public Medline (PUBMED), Scientific Eletronic Library Online (SciELO), BIREME, LILACS e Google Acadêmico. Para a busca, utilizou-se associações de descritores: resistência à insulina, nutrição, síndrome do ovário policístico, síndrome metabólica e obesidade. Os estudos demonstraram uma relação entre a SOP e DM 2, sendo considerada como fator de risco para o desenvolvimento da resistência insulínica pelas mulheres com SOP. Diversos estudos demonstraram os efeitos benéficos das catequinas (presentes no chá-verde) sobre uma ampla gama de patologias. Estudos em modelo animal concluíram que o chá-verde aumenta a sensibilidade ao hormônio da insulina por meio do aumento da atividade do transportador de glicose (GLUT4). Além disso, no Japão e na Tailândia, por exemplo, o uso de chá-verde mostrou-se eficaz como complemento de medicamentos usados no tratamento de DM 2. Concluiu-se que é fundamental a compreensão das desordens metabólicas desenvolvidas em mulheres com SOP, bem como a utilização de estratégias para a mudança no estilo de vida, por meio da adoção de hábitos alimentares saudáveis, que priorizem o consumo de alimentos que previnem o desenvolvimento das desordens metabólicas inerentes a esta síndrome.</div></div>

[STEPS]
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TITLE: iNDI PiggyBac-TO-hNGN2 transfection protocol Version 1
AUTHORS: Mark Cookson, michael.ward, Erika Lara Flores

[DESCRIPTION]
PiggyBac Method for hNGN2 transfection
Transfection protocol
Use of CEPT: Nature Methods 18, 528-541, 2021

[STEPS]
SECTION: Transfection protocol
1. Observe KOLF2.1 iPSCs under a phase contrast microscope to assess confluency and presence of cells debris. Dish should be dissociated at ~70% to 90% confluency.

Coat a well of 6 well plate to be used for transfection with 1 mL of Matrigel solution, tilting to ensure coverage of entire surface area. Place in 37 °C incubator for 30 min .



SECTION: Transfection protocol
2. Prepare E8 medium supplemented with CEPT and place in at Room temperature to warm during dissociation.

 

SECTION: Transfection protocol
3. Aspirate culture medium from well/plate that should be dissociated and wash with PBS 1X.

SECTION: Transfection protocol
4. Aspirate PBS and add half of culture volume of Accutase. 
SECTION: Transfection protocol
5. Transfer to 37 °C incubator for 10 min .

 

SECTION: Transfection protocol
6. Meanwhile aspirate Matrigel from well and add 2 mL culture medium E8 supplemented with CEPT.

SECTION: Transfection protocol
7. When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to break the colonies.

SECTION: Transfection protocol
8. Quench the Accutase adding half of the culture volume of PBS.

SECTION: Transfection protocol
9. Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.

SECTION: Transfection protocol
10. Centrifuge 5 min at 200 - 300 x g at Room temperature .
SECTION: Transfection protocol
11. Aspirate supernatant and resuspend the cell pellet in culture medium E8 supplemented with CEPT.
SECTION: Transfection protocol
12. Count cells and plate 1 - 1.5 x 106  cells into a Matrigel-coated well. Gently swirl plate to evenly distribute cells.

SECTION: Transfection protocol
13. Return plate to 37 °C incubator.
SECTION: Transfection protocol
14. After ~ 60 min to 120 min of plating cells , warm OptiMEM at Room temperature .
 




SECTION: Transfection protocol
15. Prepare one tube with:
200 µL of OptiMEM
Total of 3 µg of DNA mix in a 1:3 (Transposase:DNA) ratio
 

5 µL to 7.5 µL  of Lipofectamine Stem 
 


SECTION: Transfection protocol
16. Incubate transfection mix for 15 min to 30 min at Room temperature .

 

SECTION: Transfection protocol
17. Add transfection mix from step 15 to cells drop wise and immediately swirl the plate to evenly distribute the mix to cells.
SECTION: Transfection protocol
18. Return plate to 37 °C incubator.
SECTION: Transfection protocol
19. Next day, check for nuclear BFP positive signal (for PB-TO-hNGN2-BFP plasmid). If a positive transfection occurred and the well is confluent, re-plate the cells and puromycin select 24-48 h after transfection (it all depends on the health and transfection efficiency of the cells) with 8 µg/mL  for 2 to 14 days.

 


SECTION: Transfection protocol
20. Change media accordingly and expand cells when they reach 70-90% confluency.
TITLE: Molecular Observatory Network (MONet)
AUTHORS: maggie.bowman, Alexis Heath, Tamas Varga, Anil Battu, Will Kew, Cheng Shi, Rosey Chu, Che Clendinen, Jason.Toyoda, Odeta Qafoku, qian.zhao, izabel.stohel, Rey Hauchambe, Michael Rosenstock, Albert Lawver, nicholas.sconzo, James Anderson, Patricia Miller, andrew.townsend, Nancy Hess, John Bargar, emily.graham

[DESCRIPTION]
EMSL is leading the effort to develop a national network of environmental sampling and sensing sites to produce comprehensive molecular-level information on the composition and structure of soil, water, and resident microbial communities required to advance the span and accuracy of multiscale models of Earth systems.
Through the Molecular Observation Network (MONet), EMSL will collaborate with a broad range of partners managing an expanding network of selected natural, urban, and managed watershed, coastal, continental, and atmospheric sites, both experimental and observational. 
To establish MONet for the Biological and Environmental Research user community, research will focus on seven areas. Research focused under current development include:
Establishing the supporting field and experimental networks,
Advancing methods for model–data exchange and multiscale modeling,
Automating organic matter and soil analysis,
Developing the next generation of rhizosphere sensors and other field sensors for plants, microbes, nutrients, biomarkers, and aerosols.
The data and models generated through MONet will improve prediction of ecosystem function and response to disturbances, supporting the long-term U.S. Department of Energy goal of scientifically informed decision-making regarding the nation’s energy and environmental security and sustainability. 
For more information please visit: https://www.emsl.pnnl.gov/monet

[STEPS]
TITLE: Zebrafish (Danio rerio) Environmental Summary, LaNeC (Marabá/PA, Brazil)
AUTHORS: Caio Maximino, Monica Gomes Lima-Maximino, Diógenes Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Environmental and housing conditions experienced by laboratory animals exert profound effects on their biology, physiology, and behavior.  These parameters are important and often overlooked sources of potential variation in experiments, and should be reported in peer-reviewed publications in order to promote scientific reproducibility.  To that end, here we provide a summary of the environmental conditions in zebrafish (</span><span style = "font-style:italic;">Danio rerio</span><span>) at tanks at Laboratório de Neurociências e Comportamento 'Frederico Guilherme Graeff' (LaNeC), located in the Universidade Federal do Sul e Sudeste do Pará, Marabá/PA, Brazil. Included are data on the physico-chemical characteristics of the water, as well health, of zebrafish in separate tanks at LaNeC. </span></div><div class = "text-block">This protocol was based on Lawrence's (https://dx.doi.org/10.17504/protocols.io.mrjc54n), and is forked from that. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials:</strong><br />• Sterile PBS <br />• Anti-mouse CD3ε, clone 145-2C11 (LEAF™ format, Cat. No. 100313/100314; Ultra-LEAF™ format, Cat. No. 100339/100340) <br />• Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS and 2mM L-glutamine) <br />• Sterile, single-cell suspension (e.g., splenocytes, lymph node cells), isolated T cells or T cell subsets <br />• 96-well flat-bottom tissue culture plates with lids (e.g., Costar® Cat. No. 3596)

[STEPS]
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TITLE: Home made SM 100bp DNA ladder for agarose gel
AUTHORS: Stéphane Mauger

[DESCRIPTION]
The SM 100pb DNA Ladder is a standard size marker equivalent to the Fisher’s O'RangeRuler‱ 100 pb DNA Ladder (#SM0623). The SM 100pb Ladder allows to determine the size of double-stranded DNA fragments between 100 bp and 1500 bp and it is composed of 15 double-stranded DNA fragments of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 and 1500 bp. Like the O'RangeRuler 100 bp, the SM 100 bp gives more intense bands at 500, 1000 and 1500 bp.

At the end, the SM 100 pb DNA Ladder is 25 times cheaper than the O'RangeRuler 100 pb.

 


 



[STEPS]
SECTION: Primers and pGEM®-3Zf(+) Vectors preparation
1. Oligo primers dilution
in 16 1.5 mL microtubes, dilute 1:10 each oligo primer at 100µM to final concentration 10µM, see the SM100pb_primers.xlsx file below.
50 µL oligo primer (100µM)
450 µL nuclease free water 

Homogenize and store at 4 °C  (or at -20 °C   for a long-term storage). 

SM100pb oligo primers sequences (salt free purification): 

  

SECTION: Primers and pGEM®-3Zf(+) Vectors preparation
2. pGEM®-3Zf(+) Vectors dilution
In 1.5 mL microtube, dilute 1:100 pGEM®-3Zf(+) vector at 1µg/µL to final concentration 10ng/µL.
5 µL pGEM®-3Zf(+) (1µg/µL)
495 µL nuclease free water

Homogenize and store at 4 °C  (or at -20 °C   for a long-term storage). 

  

SECTION: PCR amplification to generate 100pb-1500pb double-stranded DNA fragments
3. PCR mix preparation
SM100pb is produced using 28 PCRs amplifications in a total of 50 µL reaction volume:
1 x 50 µL for 600pb, 700pb, 800pb, 900pb, 1100pb, 1200pb, 1300pb and 1400pb fragments
2 x 50 µL for 200pb, 300pb and 400pb fragments
3 x 50 µL for 1000pb and 1500pb fragments
4 x 50 µL for 100pb and 500pb fragments

Defreeze and vortex all reagents, except enzymes (stored at -20 °C), for approximately5 s    
 Spin down all reagents for approximately 5 s and place on ice .

In 1.5 µL microtube, prepare the PCR mix according to the following table :
 

  

SECTION: PCR amplification to generate 100pb-1500pb double-stranded DNA fragments
4. Reverse primers and mix combinaison
Defreeze and vortex all the 15 reverse oligo primers at 10µM
In a 96-well plate PCR, transfer 2 µL of each reverse oligo primers (10µM) according to the following map :
 

Vortex and spin down the PCR mix tube, transfer 48 µL in each of the 28 wells.
Seal the PCR plate.

In thermocycler, run PCR amplification with cycles follows:
 

After PCR, pool and dilute 1:2 the PCR amplification
In a 5 mL tube:
1400 mL of PCR amplification (28 x 50 µL)
420 µL  5X green GoTag buffer
980 µL nuclease free water

Homogenize and store at 4 °C  (or at -20 °C   for a long-term storage). 

Load 5 µL to 10 µL per line in a agarose gel.
TITLE: Hornwort growth media 
AUTHORS: Eftychis Frangedakis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Hornworts have a remarkable regenerative capacity. For tissue culturing / propagation you just need to transfer a small tissue fragment on fresh media and that's it... it will keep growing.</div></div>

[STEPS]
?. Using a sterile scalpel tranfer a small thallus tissue fragment (around 3mm x 3mm, but can also be smaller or bigger) on KNOP or BCD fresh media plates. Grow at 21oC, 12 h of light and 12 h of dark, 1500 lux light intensity
?. Subculture every 1-2 months
?.
TITLE: AT8 Tau Pathology Image Analysis
AUTHORS: Hemanth Ramesh Nelvagal, Toby J Curless, Zane Jaunmuktane

[DESCRIPTION]
QuPath is a bioimage analysis software designed for digital pathology and whole slide image analysis. This protocol describes how to analyse AT8 tau pathology in human brain tissue (FFPE sections with IHC).




[STEPS]
SECTION: Annotation
1. 
Manually annotate regions of interest on NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer:https://www.hamamatsu.com/us/en/product/life-science-and-medical-systems/digital-slide-scanner/U16179-01.html.
SECTION: Annotation
2. Download annotations using a Python script.


SECTION: QuPath De-Convolution and Measurements
3. Import into QuPath using a Groovy script. Refer to: Bankhead, P., Loughrey, M.B., Fernández, J.A. et al. QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017). https://doi.org/10.1038/s41598-017-17204-5
SECTION: QuPath De-Convolution and Measurements
4. In QuPath, apply colour deconvolution to distinguish DAB from the haematoxylin counterstain. 
SECTION: QuPath De-Convolution and Measurements
5. Measure the area of positive DAB staining for tau pathology using a fixed threshold value of 0.2 on the DAB deconvolved channel. 
SECTION: QuPath De-Convolution and Measurements
6. Calculate the percentage of positive DAB staining within the ROI by calculating  the area of positive
DAB staining divided by the area of the ROI and multiplied by 100.
TITLE: Agro Preparation for Mimulus in Planta Transformation
AUTHORS: Yaowu Yuan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of a </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">collection for </span><span style = ":;font-style:italic;">Mimulus in planta</span><span style = ":;"> transformation</span></a><span>. </span></div></div>

[STEPS]
?. Inoculate 4 agro colonies into separate  tubes of LB+Kan+Gent+Rif.
5 ml
?. Colony PCR from the 5 mL culture to check that it has your insert.
?. Inoculate correct agro colony into a flask of  LB+Kan+Gent+Rif, and cover with foil.
300 ml
?. Make glycerol stocks by adding  of agro to glycerol stock tube.
1 ml
?. Transfer contents of flask into 500 mL centrifuge bottles and label them.
?. Balance the bottles (use the scale and spray bottle of water to balance).
?. Centrifuge to pellet the Agro.SPEED = TIME = TEMP =
?. While centrifuging, make the resuspension solution:  total for each construct 5% sucrose =  sucrose acetosyringone = 2 mL/L =
300 ml
15 g
0 Molarity (M)
600 µl
?. After centrifuging, pour off liquid into the original flasks the Agro was grown in, and bleach the flasks.
?. Resuspend the Agro pellet in  by adding the solution to the centrifuge bottle and shaking.
[resuspension solution]
?. Pour into spray bottle for infiltration.
?. Add  to the Agro and shake to mix.
[Silwett]
?. Shake overnight at -.
?. Shake for  to  at -.
If left longer, the bacteria will begin to die.
?. Place in liquid nitrogen then store in  freezer.
?. Make acetosyringone fresh by dissolving /mL in methanol.
0 g
?. Dissolve sucrose.
?. Add acetosyringone.
?. Add dH2O to bring total volume to , and mix.
300 ml