eamag/protocols-with-steps huggingface dataset:
TITLE: Shark Tank Keto Pills
AUTHORS: https://www.scoop.it/topic/shark-tank-keto-pills-by-babelo-sar

[DESCRIPTION]
Shark Tank Keto Pills

[STEPS]
SECTION: How do Shark Tank Keto Pills Work?
1. Instant keto pills energize your body with ample energy. By consuming these instant keto pills, you quickly get to the ketosis stage, rapidly shedding excessive fats. The ketosis process creates more ketone molecules that are employed to power your cells. But only eating the keto pills will not work as you need to follow a diet routine and exercise from time to time.

Most general diets are long-drawn, tough, followed by strict rules with zero assurance of any outcome. Rest diets are severe, with added side-effects that may lead to some serious ailments. Click Here to Get Keto Instant Pills Utilize Fat for Energy with Ketosis - Boost Energy & Focus, Manage Cravings, Support Metabolism Best Keto Supplement in 2020 for Boosting Ketosis and Burning Fat Faster

But keto-diet is devoid of all those drawbacks. Using instant keto pills in a diet routine is simple and easy to follow. The instant keto pills review says that the keto pills have surprisingly no side-effects, thereby being a purely organic method of eliminating excess fats.

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Moreover, what is the differentiating factor between the normal metabolism process and ketosis metabolism process? Let us find that out. In the normal metabolic stage, glucose gets acquired by breaking down carbohydrates. Glucose is the primary fuel that runs the body. But a rise in glucose levels increases blood sugar-level that leads to toxic levels of insulin in the body that leads to diabetics. But, in the ketosis stage, fats are broken down instead of carbohydrates. So, in place of glucose, ketones switch the position to give strength and energy to the cells for optimal functioning. It usually takes three to four days to reach the ketosis stage when you follow a customized diet.
Shark Tank Keto Pills
https://www.jpost.com/promocontent/shark-tank-keto-pills-scam-exposed-2022-is-it-fake-or-trusted-700662
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TITLE: Visualization of chitin-rich tissues with Lactophenol blue slide mount
AUTHORS: Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to visualize chitin-rich tissues, such as fungal mycelium.</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: DENV Titration
AUTHORS: Fabio Gomes

[STEPS]
?. [Fixation of Cells]
Prepare fixative - 80% methanol in water
?. [Fixation of Cells]
Dump Methylcellulose overlay and blot on paper towels
?. [Fixation of Cells]
Wash gently with 1X PBS. Incubate for 10 min. Blot and dry.
?. [Fixation of Cells]
Add 0.5 mL fixative to each well. Allow plates to sit at room temperature for 10 min.
Note: Fixed cells can be stored at -70oC for future use. Leave methanol on cells when freezing.
?. [Antibody]
Wash plates with PBS. Incubate for 10 min.
If frozen plates are being used, incubate at 37oC for 30 min.
?. [Antibody]
Prepare antibody dilutions in 5% skim milk, PBS
[Skim Milk]
4G2 1:2000
?. [Antibody]
Add 200 µl to each well. Incubate at 37oC for 1 hour on a rocker.     Minimize contact with each well by maintaining contact of the pipette tip with the wall of well.
?. [Antibody]
Dump off primary antibody solution and tap plates on paper towels to remove excess solution
?. [Antibody]
Wash with 1 mL 5% skim milk, PBS
?. [Antibody]
Prepare antibody dilutions in 5% skim milk, PBS.
Anti-mouse-Per 1:2,000
?. [Antibody]
Add 200 µl to each well. Incubate at 37oC for 1 hour on a rocker.
?. [Color Development]
Dump off antibody solution. Tap on paper towels
?. [Color Development]
Wash twice with PBS. Tap on paper towel
?. [Color Development]
Add 160 µl of TrueBlue substrate per well
?. [Color Development]
Place on rocker at RT until plaques develop (10 min or longer).
?. [Color Development]
Dump off peroxidase substrate.
?. [Color Development]
Count plates or incubate at 4oC for up to 7 days.
TITLE: A Condução do Experimento - Avaliando o Jogo de Fisica
AUTHORS: Geiser Chalco Challco

[DESCRIPTION]
O protocolo corresponde ao teste de duas versões de jogo de ensino de fisica , uma fundamentada na experiencia de fluxo e uma outra sem fundamentação da teoria da experiencia de fluxo

[STEPS]
SECTION: Pre-teste
1. Todos os participantes devem assinar TCLE (se for menor de idade) ou TALE(se for maior de idade)
SECTION: Pos-Teste
4. Aplicar o FSS-2 (https://forms.gle/pjPyS168Rzv8FKQT6) e formulário de usabilidade (https://forms.gle/9s5fkfSX7LwGnyjh7) 10 min 

SECTION: Pos-Teste
4. Aplicar o FSS-2 (https://forms.gle/pjPyS168Rzv8FKQT6) e formulário de usabilidade (https://forms.gle/9s5fkfSX7LwGnyjh7) 10 min 

SECTION: Execução do Experimento
3. Os estudantes devem usar a versão sem uso de elementos da teoria de fluxo  https://fanciful-bunny-7ab9e9.netlify.app/ 45 min 

SECTION: Execução do Experimento
3. Os estudantes devem usar a versão fundamentada na teoria da experiência de fluxo (https://velvety-toffee-fbb7db.netlify.app/) 45 min 

SECTION: Pre-teste
2. Aplicar em todos os alunos os questionários DFS-2 (https://forms.gle/fvh298ac4pKCWHJL9) e de Perfil de Jogador (https://forms.gle/sxbaeUatgyoqpo3LA) 10 min 

2. Aplicar em todos os alunos os questionários DFS-2 (https://forms.gle/fvh298ac4pKCWHJL9) e de Perfil de Jogador (https://forms.gle/sxbaeUatgyoqpo3LA) 10 min
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines our alpha diversity analyses of the virome (from PHACCS) and whole metagenome (from MetaPhlan OTU table). We start by comparing the virome and whole metagenome alpha diversity values, and then look at the differences in virome and whole metagenome diversity between skin sites. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.<br /><br />

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7<br /></span>

[STEPS]
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TITLE: Optimized Protocol for RNA Extraction from Insect Samples Using TRIzol Reagent
AUTHORS: Monique da Silva Bonjour, Ian de Paula Alves Pinto, Angelo José Rinaldi

[DESCRIPTION]
RNA extraction from insect samples is a crucial step in molecular biology studies aimed at understanding gene expression, functional genomics, and various other applications. This study presents an optimized protocol for RNA extraction from insect tissues using TRIzol reagent. The protocol involves homogenizing insect samples in TRIzol, followed by phase separation with chloroform, RNA precipitation with isopropanol, and washing with ethanol. The final RNA pellet is dissolved in RNase-free water. This method ensures high yield and purity of RNA, suitable for downstream applications such as RT-PCR and RNA sequencing. The efficacy of this protocol was validated through spectrophotometric analysis and agarose gel electrophoresis, demonstrating its reliability and efficiency for extracting RNA from a wide variety of insect species

[STEPS]
SECTION: Steps
1. Cover the crucible with liquid nitrogen to keep the sample cold
SECTION: Steps
2. Place the sample (about 50mg) and macerate, keeping it cold. Remove the wings and macerate insect samples as they have a strong exoskeleton and the presence of chitin
SECTION: Steps
5. Add 1mL of Trizol and wait for it to thaw
SECTION: Steps
3. Mix the sample with Trizol in the crucible and transfer to a 1.5 mL microtube
SECTION: Steps
4. Add 200 µL of ice-cold chloroform for phase separation. Shake the tube vigorously by hand for a full 15 seconds. Let sit at room temperature for 2-3 minutes

NOTE: You should see the two mixtures separating almost immediately: pink on the bottom and clear on top
SECTION: Steps
6. Centrifuge at 12000 rpm for 15 min at 4° C

NOTE: Any denatured proteins might appear as a white interface. Avoiding this interface, carefully move the clear top liquid to a new clean, labeled tube
SECTION: Steps
7. Remove 500 µL of supernatant and add 400 µL of ice-cold isopropanol to precipitate the RNA (always 100 µL less)
SECTION: Steps
8. Leave 10 minutes at room temperature

NOTE: Leaving to precipitate in the -20°C is not recommended because it might cause contaminants to co-precipitate
SECTION: Steps
9. Centrifuge at 12000 rpm for 10 min 4° C 

NOTE: You may see the pellet as a clear, gel-like ball at the base of the tube, or a small white smear
SECTION: Steps
10. Remove the isopropanol and wash with 1 mL of 75% ethanol
SECTION: Steps
11. Centrifuge at 9500 rpm for 5 min at 4° C
SECTION: Steps
12. Discard the alcohol and wait for the pellet to dry for 5 to 10 minutes at room temperature

NOTE: Remove as much of the ethanol as possible    
SECTION: Steps
13. Resuspend in 30 µL of RNAse-free water depending on the size of the pellet formed
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The concentration and purification of giant DNA viruses from bacteria-containing cultures can present problems because the particle size of giant viruses overlaps with that of smaller bacteria, hence filtration as the only separation method is not possible.</p>
<p>In this protocol, a combination of centrifugation, tangential flow filtration, and density-gradient ultracentrifugation is used to concentrate and purify particles of the giant marine virus CroV that infects the heterotrophic flagellate <em>Cafeteria roenbergensis</em>.</p>

[STEPS]
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TITLE: Preparing mitochondrial samples for immunoblot analysis
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for preparation of mitochondrial samples for immunoblot analysis.

[STEPS]
1. Thaw mitochondrial stocks on ice, and aliquot out the desired amount of mitochondria.
2. Centrifuge each aliquot for 10 min  10000 x g, 4 °C 

3. Carefully aspirate the supernatant from each sample. 
4. Add a volume of 1x SDS sample buffer (5% w/v SDS, 10% v/v glycerol, 100 mM DTT, 50 mM Tris-Cl pH 6.8) equal to the amount of mitochondria (in ug) to each sample. Eg. If each sample contains 20 µg of mitochondria, add20 µL of 1x SDS sample buffer.

5. Vortex samples for ~5 seconds to mix, and then boil at 99 °C with shaking at max speed for 10 min. 

6. Allow samples to cool to room temperature, quickly centrifuge to collect liquid to the bottom of the tube, and vortex for ~3 seconds to ensure the samples are homogenous. 
7. Samples can now be directly loaded onto an SDS-PAGE gel, or stored at -20 °C for later use.
TITLE: Antibody and TDP-43 RNA aptamer dual staining to detect patterns of  co-pathology in FFPE-preserved human tissue, as described in Rifai et al., 2024 (Brain Pathology): A SOP and tick-sheet.
AUTHORS: Fergal M Waldron, Olivia Rifai, Jenna Gregory

[DESCRIPTION]
Here we provide a SOP to outline the correct procedures to dual stain FFPE human tissue for, 1) pathological TDP-43 protein, along with 2) any other protein of interest, with the aim of visualising protein of interest histology in the context of TDP-43 pathology.
 
This protocol can be implemented for dual staining of any rabbit or mouse primary detection antibody (using the Novolink Polymer Detection System), with our TDP-43 RNA aptamer (biotinylated) as published in Acta
Neuropathologica here https://link.springer.com/article/10.1007/s00401-024-02705-1 in Spence and Waldron et al., 2024. 
 
The resulting dual stain will show brown chromogen (detecting the target of the primary antibody) with red chromogen (detecting TDP-43 pathology with our TDP-43 RNA aptamer.

Reference for citations of this method

Clinicopathological analysis of NEK1 variants in amyotrophic lateral sclerosis.
Olivia M. Rifai, Fergal M. Waldron, Danah Sleibi, Judi O’Shaughnessy, Danielle J. Leighton*, Jenna M. Gregory*‡ (2024). Brain Pathology doi: 10.1111/bpa.13287  (in press at the time of publication of this SOP) *equal contributions, ‡corresponding author.

Antibody and TDP-43 RNA aptamer dual staining to detect patterns of  co-pathology in FFPE-preserved human tissue, as described in Rifai et al., 2024 (Brain Pathology): An SOP and tick-sheet.
Fergal M. Waldron, Olivia M. Rifai, Jenna M. Gregory‡, (2024). Protocols.io 2024; doi: tbc
‡corresponding author

[GUIDELINES]
See SOP pdf.

[STEPS]
1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In vitro transcriptions assay for cyanobacterial RNA polymerase. This protocol can be used either to do an in vitro transcription with small DNA fragments with desired promoter region or to use it with a promoter region integrated in a plasmid.</p>

[BEFORE_START]
<p>Prepare all Buffer, measure RNAP and DNA concentrations</p>

[GUIDELINES]
<p>RNA experiments! Always wear gloves and prepare probes under RNAse free and cooled conditions!</p>

[STEPS]
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TITLE: Quick Ligation Protocol (M2200)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols is to be performed with the Quick Ligation Reaction Buffer. Please see the NEB website for more information.</div></div>

[STEPS]
?. Set up the following reaction in a microcentrifuge tube .  AB1COMPONENT20 μl REACTION2Quick Ligase Reaction Buffer (2X)*10 µl3Vector DNA (3 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free Waterto 20 µl6Quick Ligase1 µl
on ice
Quick Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
AB1COMPONENT20 μl REACTION2Quick Ligase Reaction Buffer (2X)*10 µl3Vector DNA (3 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free Waterto 20 µl6Quick Ligase1 µl
?. Gently mix the reaction by pipetting up and down and microfuge briefly.
?. Incubate at  () for .
0 Room temperature
25 °C
?. Chill  and transform  –  of the reaction into  competent cells. Alternatively, Store at .
on ice
1 µl
5 µl
50 µl
-20 °C
Do not heat inactivate – heat inactivation dramatically reduces transformation efficiency.
TITLE: Protein Purification strep-tag FPLC 
AUTHORS: Cleo B.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein purification of proteins containing a strep-tag using FPLC. </div><div class = "text-block">Necessary: </div><div class = "text-block">Have performed Protein expression using E. coli strain BL21DE3</div><div class = "text-block">To continue see: Protein gel sample preparation V2. </div></div>

[STEPS]
?. [Buffers]
1 L Buffer W: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTAFilter this buffer with a 22 um filter 
100 mL Buffer E: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTA2.5 mM desthiobiotin Filter this buffer with a 22 um filter 100 mL Buffer E: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTA2.5 mM desthiobiotin 1L 0.5 M NaOH (filtered with 22 um filter) 1L MQ (filtered with 22 um filter)
?. [Protein extraction ]
Place post-induction culture from Protein expression using E. coli strain BL21DE3 on ice. For FPLC have 2 L of cell culture.
?. [Protein extraction ]
Centrifuge culture at 5000xg for  at
4 °C
?. [Protein extraction ]
The pellet is resuspended in  buffer W per 100 ml cell culture containing one crushed cOmplete mini tablet for 2 L cell culture.
1 ml
?. [Protein extraction ]
The cells are sonificated (VS70 T rod, 25% 1 sec on 2 sec off for . On ice water). For small amounts use the MS72 rod. From this point be very sure to keep the cells on ice as much as possible.
?. [Protein extraction ]
The cell extract is centrifuged for  at 30 000 g.
?. [Protein extraction ]
The supernatant is collected and filtered first with 0.45 um filter and then with 0,22 um filter. If there are small sample volumes: add  buffer W.
3 ml
?. [Protein extraction ]
?. [FPLC ]
Connect computer to system
?. [FPLC ]
Set a manual alarm specific for the column you're using All speeds are dependent on the machine/column that is used
?. [FPLC ]
Wash all pumps with MQ
?. [FPLC ]
Wash the system with MQ (~20 mL)
?. [FPLC ]
Set valve position to waste
?. [FPLC ]
Connect column wet (MQ) with a low flow rate (~1 ml/min) Wash column with MQ Increase flow rate slowly
?. [FPLC ]
Equilibriate column with buffer WSlowly increase flow rate Wait until the conductivity and UV have stabilised
?. [FPLC ]
Wash peristaltic pump with 15 mL MQ Introduce cell lysate in column using the peristaltic pump (connect wet) Story flow through and a bit of lysed cells
?. [FPLC ]
Wash the column with buffer W until the lines stabilize
?. [FPLC ]
Start elution with buffer E Collect all the fractions per 1/1.5 mL Wash until the protein peak has passed Place all the fractions on ice immediately Fractions can be placed on analytical SDS-page gels
?. [FPLC ]
Wash the column with MQ (3  CV) Wash the column with NaOH (3 CV)Wash the column with ethanol (decrease speed before changing to ethanol) (3CV) Wash the system with ethanol
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The major ion concentration data of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), chloride (Cl), sulphate (SO<sub>4</sub>), bicarbonate (HCO<sub>3</sub>), carbonate (CO<sub>3</sub>) and pH (if it was coupled with ion data) were input into the database. The data were drawn from a large geographic scale across Eurasia (Austria, China, Hungary, Kazakhstan, Mongolia, Russia, Serbia, Turkey) and from a large number of saline lakes and pans (N=220) with minimum a 1.0 g L<sup>–1</sup> salinity threshold. The 1.0 g L<sup>–1</sup> salinity threshold was selected based on a former study (Boros et al., 2014), where this threshold was experimentally found to be the characteristic boundary of soda ecosystems. Salinity was estimated by the sum of measured concentrations of eight major ions (Na, K, Ca, Mg, Cl, SO<sub>4</sub>, HCO<sub>3</sub>, CO<sub>3</sub>). As generally known, sodium is by far the most common cation in saline lakes and necessarily the dominant cation in soda type lakes. Therefore, sites were excluded from the dataset if Na was not the most abundant ion. If seasonal or annual water data were available, mean values were put into database. Most of the data came from papers (sources are indicated in the table).</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Anti-Neu5Gc may be used for staining cells prior to analysis by Flow Cytometry. This kit contains all the essential components needed to identify Neu5Gc on the surface of cells by flow cytometry.</p>
<p> </p>
<p>Use of the blocking agent (Neu5Gc Assay Blocking Solution) provided in the kit is essential, as commonly used blocking agents invariably contain serum, or serum components, that can either inhibit detection or introduce Neu5Gc contamination.</p>
<p> </p>
<p>Tissue culture-grown CHO-K1 can be used as a positive control, and human peripheral blood mononuclear cells serve as a negative control. Adherent tissue culture-grown cells should be released from the culture flasks by using 5-10mM EDTA for 10 minutes at room temperature. Other non-enzymatic methods, such as Accutase, may be used. Immediately wash cells in blocking buffer that contains a lower concentration of EDTA, and resuspend cells in blocking buffer to determine cell numbers and viability.</p>

[GUIDELINES]
<p>It is assumed that the user is familiar with the general principles and practices of Flow Cytometry.</p>
<p> </p>
<p><strong>Items required but not supplied:</strong></p>
<ul>
<li>Reagent grade ultra-filtered water</li>
<li>Disposable cytometry tubes</li>
<li>Positive Control</li>
<li>Fluorescently-conjugated Secondary Antibody</li>
<li>Buffer</li>
<li>PBS</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em></em>The light/dark test is increasingly being adopted as one of the main behavioral assays in zebrafish neuroscience research. It is based on the innate preference of adult zebrafish for a black vs. a white compartment. Modifications and extensions of the protocol increased its breadth, but also its heterogeneity. The protocol presented here involves confining the animal in a central compartment for a initial 3-min acclimation period, followed by free exploration of an acryllic tank with a black and a white compartment. In addition to observing the spatial distribution of the animal, "ethological" variables (risk assessment, erratic swimming, freezing, thigmotaxis) are also manually scored in the white conpartment; these endpoints are selectively sensitive to drug treatments. The protocol is intended to measure anxiety-like behavior, with anxiogenic effects decreasing time on white and increasing risk assessment and thigmotaxis; some treatments also increase erratic swimming and freezing.</p>

[BEFORE_START]
<ol>
<li>Room illumination is a critical variable, as it can alter fish behavior and the aversiveness of the white compartment. Before each trial, light levels should be measured; variations above or below 2 SDs from the average should be considered as too discrepant for an experiment. Ideally, lighting above the tank should be on the range of 500-600 lux.</li>
<li>Use Gaussian white noise throughout the experiment to cover unwarranted noise in the experimental room.</li>
<li>Bring the animal in a temporary transport recipient into the behavioral test room.</li>
</ol>

[GUIDELINES]
<p>Standard guidelines for the ethical care of fish in research apply. Before beggining experiments, researchers should obtain all legally binding licenses, inclusing IACUC approval.</p>

[STEPS]
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