eamag/protocols-with-steps huggingface dataset:
TITLE: Fast rodent genotyping
AUTHORS: Alexandros C Kokotos, Timothy A. Ryan

[DESCRIPTION]
This protocol describes a very fast method (approximately 2 h) to do genotyping from rodent tail clips and other samples.

[BEFORE_START]
The current protocol describes genotyping using rodent tail clips. This protocol has been verified using both rats (Sprague Dawley strain) and mice (C57BL/6J strain). It can also be applied to other types of biological samples, like blood, buccal swabs etc.
Example genotyping applied includes PARK20 mice and sexing of wt rats.
Operating scissors should be kept very sharp at all times, by cutting through aluminum foil or stainless steel sponges.

[STEPS]
SECTION: DNA extraction
1. Tail clips from mice or rats of appropriate age should be snipped using sharp operating scissors and in compliance to local protocols by national and institutional regulatory organizations and placed in sterile Eppendorf tubes.
SECTION: DNA extraction
2. Note: This protocol has been tested using the smallest tail clips possible of ~ 1 mm. Always clean the scissors with 70 % (v/v) Ethanol in between animals.
SECTION: PCR
7. Prepare PCR reactions at Room temperature as follows
Note: Preparing a master mix is suggested for several samples.
 
 



SECTION: DNA extraction
3. Add 20 µL of Lysis Buffer and 0.6 µL of Proteinase K in each sample tube.
Note: Preparing a master mix is suggested for several samples.
SECTION: DNA extraction
4. Incubate samples at 95 Room temperature for 1 min.
SECTION: DNA extraction
5. Put samples in the heat block at 98 °C for 1 min.

SECTION: DNA extraction
6. Spin down the samples using a mini-centrifuge for 30 s and transfer lysate to new tubes.

SECTION: PCR
8. Note: PCR reaction may need optimization. Primers should be used at a final concentration of 0.2 micromolar (µM). In this example, 0.4 µL is added for each of two primers from a 10 micromolar (µM) stock.

SECTION: PCR
9. Place PCR samples in a PCR thermocycler. Set up the thermocycler with the following program using a Hot Start function.
  
 
 

 
SECTION: DNA gel electrophoresis
11. During the PCR, prepare an appropriate agarose percentage DNA gel.
SECTION: PCR
10. Note: PCR program may need optimization.
SECTION: DNA gel electrophoresis
12. Load PCR sampes along with an appropriate DNA ladder on the DNA gel.
SECTION: DNA gel electrophoresis
13. Submit the DNA gel to electrophoresis.
SECTION: DNA gel electrophoresis
14. Visualize gel using an appropriate device.
TITLE: Cell-based analysis of PINK1-Parkin pathway activation in primary mouse  cortical neurons
AUTHORS: Odetta Antico, Miratul M. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mutations in PINK1 cause early-onset Parkinson’s disease. PINK1 becomes stabilised and active upon mitochondrial depolarisation. This leads to phosphorylation of ubiquitin and Parkin via Serine 65 residues and a feed forward mechanism whereby PINK1 phosphorylates newly formed polyubiquitin chains, generating phospho-ubiquitin, which further promotes Parkin recruitment and activation. Once activated, Parkin ubiquitylates proteins at the outer face of the outer mitochondrial membrane (OMM) and then initiates a downstream pathway that eventually leads to mitophagy, a mitochondria-specific type of autophagy. Notably, much of previous investigation into PINK1/Parkin activity has been performed in non-neuronal human cancer cells where Parkin and/or PINK1 is over-expressed. Here we report a protocol for generation of mouse embryonic cortical neuronal cultures that produce high cell yields and can be used for studying endogenous PINK1 and Parkin signalling by biochemical methods and proteomics.</div></div>

[STEPS]
TITLE: Chimeric Protein-LG and Protein-LAG sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA is used to study the interactions between protein-LG (SpLG) and protein-LAG (PLAG) with different immunoglobulin preparations from mammals and avian species.</div></div>

[STEPS]
?. This ELISA is used to study the interactions between protein-LG (SpLG) and protein-LAG (PLAG) with different immunoglobulin preparations from mammals and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpLG 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 PLAG 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 0.32.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for quantification of circulating microRNA using TaqMan Low Density Array.</p>
<p>MicroRNAs are purified from platelet-poor plasma using Nucleospin columns</p>
<p>During the purification step samples are spiked with cel-miR-39 as a mean of technical normalization</p>
<p>Normalization is performed using the ΔCt-method</p>

[BEFORE_START]
<p>We recommend to test if the volume of spike-in added during microRNA purification is adequate before purifing all your samples.</p>
<p>When testing the preamplification product, the Ct-values of the spike-in and the target miRNA should be within the same range, otherwise adjust the volume of spike-in added during miRNA purification.</p>

[GUIDELINES]
<p>Blood samples should be obtained using a minimum of venous stasis and with discard of the first 3 mL of blood.</p>
<p>Platelet-poor plasma should be prepared within 2 hours from blood sampling</p>
<p> </p>

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TITLE: tetA dual selection protocols  
AUTHORS: Carolyn N Bayer, Ana Gabriela Veiga Sepulchro, Maja Rennig, Morten Norholm

[DESCRIPTION]
Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit bacterial chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for creation of minimized genomes. Genetic tools are needed to select for cells that underwent editing, and dual selection markers that enable both positive and negative selection are highly useful. Here we present an optimized and easy-to-use version of thetetAdual selection marker and demonstrate how this tetAOPT can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetAOPT can be used to create scarless knockouts across the E. coli genome with an efficiency above 90% whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the expression level, we show that tetAOPT enables advanced genome engineering of both genetranslation and transcription. Finally, we demonstrate the use of tetAOPT for genome engineering in the industrially relevant probiotic strain E. coli Nissle.

[STEPS]
TITLE: Bacterial Delivery System
AUTHORS: contact.microbialtec  

[DESCRIPTION]
Protein synthesis can be controlled with the bacterial translation machinery. Control of invasion is necessary to carry the produced proteins into cells. Invasion requires both flagella and the type III secretion system-1 (T3SS-1). Flagella are essential for cell invasion because they sense the cell surface and determine the optimal location for invasion32. T3SS-1 is a needle apparatus that initiates invasion by injecting effector proteins into cells. These proteins rearrange the actin cytoskeleton and induce endocytosis of the bacteria. Production of these two bacterial structures is controlled by the factors fliZ and hilD, which are, in turn, controlled by the master regulator flhDC. Protein release requires the activation of bacterial genes specifically inside cells. InSalmonella, this can be controlled with the promoters of SPI2 genes.

[STEPS]
TITLE: The TARGET System: Rapid Identification of Direct Targets of Transcription Factors by Gene Regulation in Plant Cells
AUTHORS: Matthew D. Brooks, Kelsey M. Reed, Gabriel Krouk, Gloria M. Coruzzi, Bastiaan O. R. Bargmann

[DESCRIPTION]
The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, makes this plant cell-based approach a valuable tool for a higher through-put approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.

NB. The original uploaded pdf contained a typo; PEG solution contains 0.4 M mannitol (not 0.4 mM).

[STEPS]
TITLE: Growing bacteria in b-broth
AUTHORS: Cristian Riccio

[STEPS]
?. Find an isolated colony of bacteria on the agar plate.
?. At the Bunsen burner, touch the colony with the tip of a pipette tip and drop the pipette tip into a 100 ml bottle of b-broth.
?. Put the 100 ml bottle of b-broth with the bacteria in the incubator, 37 C for E. coli.
TITLE: ColabFold Protein Structure Prediction and Membrane Interaction Modeling
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
ColabFold Protein Structure Prediction and Membrane Interaction Modeling

[STEPS]
1. Protein structures (WT and phospho-mutant α-syn) were predicted using ColabFold (https://colabfold.mmseqs.com/),which uses MMseqs2 for homology detection and multiple sequence alignment, and AlphaFold2 for simulated protein folding.
2. The full-length  human α-synuclein isoform 1 sequence (Uniprot, P37840-1) was input into ColabFold with 3 recycles, and the model with the highest overall IDDT was chosen as representative. For modeling of the phospho-mimetic mutant of α-syn, Ser129 was replaced with an aspartate residue before input.
3. To model the interaction between α-syn and SV-membranes, an all-atom simulation was set up, which included  the previously selected ColabFold protein models and a phospholipid bilayer made up of 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) spread in the x-y plane. The protein models were introduced as close to the membrane as possible, while ensuring no disruption of the bilayer, and then the simulation box   was solvated with TIP3P water molecules, and charge neutralized by the required number of K+ and Cl- ions.
4. Simulations      were      conducted      using      GROMACS      v.2021.3      [https://www.gromacs.org/(RRID:SCR_014565)] [doi: 10.5281/zenodo.5053201] with a CHARMM36 force field [(RRID:SCR_014892) https://www.charmm.org/archive/charmm/resources/charmm-force-fields/]at  a temperature  of  310.15K.
5. Particle size of the simulations was 618946 and 324399 for wild-type and S129D α-synuclein, respectively. After the simulation setup, energy minimization was performed using the steepest descent minimization algorithm, and the LINCS algorithm [(RRID:SCR_016486) http://www.lincsproject.org/] was used to achieve the constraints for hydrogen bonds.
6.  Equilibration of the system was performed with two steps of NVT with Berendsen  temperature coupling used for temperature correction with a time step of 1 femtosecond, followed by four steps of NPT with Berendsen temperature and pressure coupling with a time-step of 1 femtosecond for the first NPT step and 2 femtoseconds for the following three.
7. All equilibration steps were run for 125 picoseconds each.The molecular dynamics production run was performed, without any constraints, for approximately 200ns using Nosé-Hoover temperature coupling  and Parrinello-Rahman pressure coupling, with a time-step of 2 femtoseconds.
8. In the final model, the helicity of each residue is represented as a fraction of time spent either as a component of an α-helical segment of the protein, a 1, or otherwise, a 0. The depth profile of each protein model is represented as the position of each residue with respect to the phosphate head group and is shown as the mean and standard deviation of the last 20 nanoseconds of the production run.
TITLE: DFP-induced mitophagy experiments
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the DFP-induced mitophagy experiments.

[STEPS]
SECTION: DFP-induced mitophagy
1. To induce mitophagy, 

Treat the cells for 1440 min with 1 millimolar (mM) Deferiprone (DFP) (379409, Sigma Aldrich), an iron chelator that mimics hypoxic conditions through stabilization of the transcription factor HIF1α and subsequent upregulation of NIX and BNIP3. 
SECTION: DFP-induced mitophagy
2. Freshly dissolve the compound in sterile dH2O on the day itself and boil for 1 min at 95 °C to dissolve the compound.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Fixation of mouse tissues for either histology or fluorescence microscopy is an essential first step in tissue preparation. In this protocol, we outline a reliable method for fixation and perfusion of mice for tissue histology or microscopy. Tissues prepared by this method can be used downstream as FFPE sections, cryosections for fluorescence microscopy, or tissue clearing for whole tissue imaging. For mice with transgenic fluorescent reporters, it is especially important that the tissues are preserved properly to ensure that the signal of the fluorescent reporter can be imaged. 

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol seeks to provide all information needed to allow regular spawning of Aiptasia and safe handling of the spawn.</p>
<p> </p>
<p>It is based on the protocol described in Grawunder et al., 2015.</p>

[GUIDELINES]
<p>This protocol refers to three different sizes of polycarbonate plastic food containers to maintain Aiptasia in:</p>
<p>     -<strong>small (250 ml)</strong>:  Cambro 92CW</p>
<p>     -<strong>medium (750 ml)</strong>: Cambro 62CW</p>
<p>     -<strong>large (2000 ml)</strong>: Cambro 44CW</p>
<p> </p>
<p>The artificial seawater (ASW) used in the Pringle lab is Coral Pro Salt (manufactured by Red Sea) dissolved in de-ionized water to a salinity of between 32 and 34 (usually ~37.4 g/l).</p>
<p> </p>
<p>The Pringle lab uses the male, clonal Aiptasia strain CC7 to obtain sperm and the clonal female strains H2, PLF3/F003, PLF5 and used also PLF8 in the past to produce eggs. All strain combinations yield viable planula larvae.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for human intestine cell dissociation. </p>

[BEFORE_START]
<div title="Page 1">
<p><strong>Checklist prior to beginning:</strong><br />-Centrifuges, large and small, set to 4 C<br />-Make enzyme stock; place 2 tubes of enzyme on dry ice.<br />-Make 0.01% BSA/PBS (50 mL)<br />-Things you need: petri dishes, clean forceps, razor blade, pipets, 30 μM filters, timer.</p>
<p> </p>
<p><strong>Stock solution for enzyme </strong></p>
<ul>
<li>895 μL DPBS</li>
<li>5 μL 0.5 M EDTA (2.5 mM final)</li>
</ul>
<p>→Add 100 μL enzyme (100 mg/mL) to 900 μL of enzyme stock to make 1X enzyme mix. Add 28 mg of tissue to each 900 μL of enzyme mix.</p>
</div>

[STEPS]
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TITLE: Traditional Chinese Medicine as add-on treatment of triple/quadruple therapy for Helicobacter pylori infection: an overview of systematic reviews and meta-analyses (Protocol)
AUTHORS: Xiaobei Si, Yu Lan

[DESCRIPTION]
Background: In recent years, many studies reported the anti-Hp effects of Traditional Chinese Medicine. Based on these studies, there had been many relevant systemic reviews and meta analyses with variable results Aim: This overview aims
to investigate the general characteristics and assess the methodological quality of these systemic reviews/meta ana lyses (SR/MAs). Method: In this overview, we intend to includ systemic reviews/meta analyses with comparison of Chinese medicine and western medicine on helicobacter pylori infection by searching databases of Medline, Embase, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang Database until December 19th, 2023. AMSTAR 2 and GRADE system tools will be adopted to assess the methodological quality and evidence quality characteristics. Conclusion: The methodological
quality of these SR/MAs will be assessed as clinical evidence and our result may help further use of them. 

[STEPS]
1.
TITLE: Combining long-lasting insecticidal nets and indoor residual spraying for malaria prevention in Ethiopia: study protocol for a cluster randomized controlled trial
AUTHORS: Bernt Lindtjorn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background: Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the main malaria prevention interventions in Ethiopia. There is conflicting evidence that the combined application of both interventions is better than either LLINs or IRS used alone. This trial aims to investigate whether the combination of LLINs (PermaNet 2.0, Vestergaard Frandsen, Lausanne, Switzerland) with IRS using propoxur will enhance the protective benefits and cost-effectiveness of the interventions against malaria and its effect on mosquito behavior, as compared to each intervention alone.
Methods/Design: This 2 x 2 factorial cluster randomized controlled trial is being carried out in the Adami Tullu district in south-central Ethiopia for about 116 weeks from September 2014 to December 2016. The trial is based on four arms: LLINs + IRS, LLINs alone, IRS alone and control. Villages (or clusters) will be the unit of randomization. The sample size includes 44 clusters per arm, with each cluster comprised of approximately 35 households (about 175 people). Prior to intervention, all households in the LLINs + IRS and LLINs alone arms will be provided with LLINs free of charge. Households in the LLINs + IRS and IRS alone arms will be sprayed with carbamate propoxur once a year just before the main malaria transmission season throughout the investigation. The primary outcome of this trial will be a malaria incidence based on the results of the rapid diagnostic tests in patients with a fever or history of fever attending health posts by passive case detection. Community-based surveys will be conducted each year to assess anemia among children 5–59 months old. In addition, community-based malaria prevalence surveys will be conducted each year on a representative sample of households during the main transmission season. The cost-effectiveness of the interventions and entomological studies will be simultaneously conducted. Analysis will be based on an intention-to-treat principle.
Discussion: This trial aims to provide evidence on the combined use of LLINs and IRS for malaria prevention by answering the following research questions: Can the combined use of LLINs and IRS significantly reduce the incidence of malaria compared with the use of either LLINs or IRS alone? And is the reduced incidence justifiable compared to the added costs? Will the combined use of LLINs and IRS reduce vector density, infection, longevity and the entomological inoculation rate? These data are crucial in order to maximize the impact of vector control interventions on the morbidity and mortality of malaria.
Trial registration: PACTR201411000882128 (8 September 2014).</div></div>

[STEPS]
TITLE: Phosphate Buffered Saline (PBS)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare Phosphate Buffered Saline (PBS). PBS (-) and PBS (+) which includes Tween are used as biologically neutral wash solutions in many processes including In Situ Hybridization (ISH), Immunohistochemistry (IHC) and Fixation.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Collection of Protocols and Guidelines for Phase 3 study of Vaccine Candidate for COVID-19
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a collection of protocols for: "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]