eamag/protocols-with-steps huggingface dataset:
TITLE: Population relationships of Oomyzus scaposus (Hymenoptera: Eulophidae)
AUTHORS: nedved, Lida Fekrat

[DESCRIPTION]
Comparison of COI DNA barcodes from Iranian and other populations of Oomyzus scaposus and other species of Oomyzus. 

[STEPS]
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
1. We extracted DNA from the wasps using Tissue DNA genomic DNA extraction mini kit (Favorgen) according to the manufacturer’s instructions. 
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
2. Partial sequences of the mitochondrial gene Cytochrome Oxidase I (COI) were amplified using the primers HCO and LCO (Folmer et al., 1994). PCR cycling conditions consisted of an initial denaturation of 3 min at 94°C followed by 35 cycles, each of 30 s at 94°C, 40 s at 52°C and 30 s at 72°C, and a final extension of 72°C for 10 minutes. After a check through electrophoresis, all the PCR products were sequenced by BioneerCorporation (Korea). 
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
3. Forward and reverse sequences were assembled using Geneious, version 2019. 
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
4. Sequence similarities were checked using the BLAST program of the GenBank database.
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
5. The sequences were submitted to GenBank under accession numbers OP784016 and OP784017. All specimens used for sequencing are represented by voucher specimens belonging to the same populations and collected at the same time from the same host and are deposited in the private collection of the first author (AB). For phylogenetic analysis, 15 COI sequences of Oomyzus species available in the GenBank database (NCBI) were retrieved (see accession numbers in Fig. 3). A parasitoid wasp, Baryscapus, from another genus of the same subfamily (Tetrastichinae) was included as an outgroup for phylogenetic analysis. 
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
6. Phylogenetic relationships were reconstructed using MEGAX with the Kimura 2-parameter (K2) distance model, and the Maximum Likelihood (ML) method.One thousand bootstrap replicates were performed to assess branch support in the given tree.     
SECTION: Population relationships of Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae)
7. Pairwise Kimura-2-parameter (K2P) distance was measured to understand the evolutionary divergence rate among the species based on the COI gene – estimating the pairwise intra- and inter-species K2P distances among the sequences.
TITLE: Cylinder behavior test
AUTHORS: Hong-Yuan Chu

[DESCRIPTION]
This protocol describes cylinder behavior test.

[STEPS]
SECTION: Procedure
1. Move the test mice from their housing room to the testing room and allow to acclimate for around 120 min.
SECTION: Procedure
2. Keep the test environment under dim white-light illumination (around 150 lux).
SECTION: Procedure
3. Place two mirrors perpendicular to each other to form a ninety-degree angle.
SECTION: Procedure
4. Clean a 600-ml glass beaker with 70% ethanol and water before the test.
SECTION: Procedure
5. Place the 600-ml glass beaker in front of the two mirrors.
SECTION: Procedure
6. Place a digital camcorder in front of the 600-ml glass beaker towards the glass beaker and mirrors. 
 

SECTION: Procedure
7. Place a test mouse into the 600-ml glass beaker and start recording.
SECTION: Procedure
8. After 10 minutes, stop recording and return the test mouse immediately to its home cage.
SECTION: Procedure
9. Clean the 600-ml glass beaker with 70% ethanol and water.
SECTION: Analysis
10. Analyze videos offline by a person blinded to the treatments.
SECTION: Analysis
11. Give a score of “left forepaw”, “right forepaw”, or “both” based on which paw was used during a rear.
If the animal walked up the wall during a rear, that counted as “both”. At the end, “both” was divided equally between “left paw” and “right paw”.
Only one point was given per rear. Another point was not given until the animal returned to all fours and reared again. 
The paw must be splayed out and pressed against the glass cylinder to count as a point – resting the paw gently does not count. 

SECTION: Analysis
12. Sum up the total number of touches for each forepaw and determine the percentage used by dividing the number of touches for each forepaw by the total number of touches.
TITLE: Preparation of acid-washed glass coverslips for immunofluorescence microscopy
AUTHORS: Jenna Ekstrom

[DESCRIPTION]
Rizzardi Lab (Adapted from Schwer Lab- UCSF)

[STEPS]
1. Add coverslips to 10cm culture dish
2. Add ~20 mL of 1 N HCl; swirl on orbital shaker for 2 h at RT.Room temperature 

3. Remove acid, rinse with 2 x 25 mL PBS until pH is at least 6.0.
4. Rinse 1 x 25 mL water.
5. Add ~20 mL of 70% EtOH; swirl on orbital shaker for 10 min, RT.Room temperature 

6. Remove ethanol, transfer coverslips to Whatman paper. Let dry in TC hood.
7. Sterilize coverslips before use
7.1. Option 1:
Transfer to glass beaker, cover with aluminum foil and autoclave (dry cycle, 30 min sterilization time)
7.2. Option 2:
Transfer to new sterile 10cm culture dish and turn on UV.  Leave in hood until ready for use.
TITLE: Preparation of 1L of Nematode Growth Medium (NGM)
AUTHORS: Alfonso  Pérez Escudero, gabrielmadirolas, Alid Al-Asmar

[DESCRIPTION]
Original source of the protocol: WormBook Methods http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html.
This protocol is for making Nematode Growth Medium (NGM), a standard agar-based medium to grow C. elegans in petri dishes. It describes both medium preparation and pouring. 

[STEPS]
1. In order to perform this protocol, you will need the following solutions:
 5mg/mL cholesterol in absolute ethanol (do not autoclave) (stored in freezer)
pH=6 phosphate buffer (as prepared in: https://www.protocols.io/view/preparation-of-0-5l-of-phosphate-buffer-ph-6-0-n2bvj8r7bgk5/v1)
1M magnesium sulfate (as prepared in: https://www.protocols.io/view/preparation-of-1m-magnesium-sulfate-solution-mgso4-ca4rsgv6)
1M calcium chloride (as prepared in: https://www.protocols.io/view/preparation-of-1m-calcium-chloride-solution-cacl2-b8parvie)
2. Add 16 g of agar powder to a clean 1L bottle.

3. Add 2.32 g of sodium chloride (NaCl). 
4. Add 2 g of peptone.

5. Add 780 mL of milliQ water.

6. Add a clean magnet (for stirring) and autoclave.
8. While the mix is being stirred, do the following steps in that order, dispensing the reagents right above the surface (to avoid bubbles) with a sterile pipette: 
9. Add 20 mL of phosphate buffer as prepared in https://www.protocols.io/view/preparation-of-0-5l-of-phosphate-buffer-ph-6-0-n2bvj8r7bgk5/v1. This should be added first to prevent any unnecessary precipitation of the next reagents. Wait for ~20 seconds after adding the buffer to give it time to mix well and stabilize the pH.
10. Add 0.8 mL of 1M magnesium sulfate (MgSO4) solution as prepared in https://www.protocols.io/view/preparation-of-1m-magnesium-sulfate-solution-mgso4-ca4rsgv6. 

11. For this step only, you can put the pipette right below the surface of the agar, otherwise cholesterol tends to stick to the surface.
Add 0.8 mL of cholesterol (5mg/mL in ethanol, stored in freezer).
12. Add 0.8 mL of 1M calcium chloride (CaCl) solution as prepared in https://www.protocols.io/view/preparation-of-1m-calcium-chloride-solution-cacl2-b8parvie.
This step should make the medium a bit cloudier, but remain translucent. 
13. Pour in petri dishes before it solidifies, using a pipette and trying to have the same volume in all dishes. We usually pour the plates on the bench, but to minimize the chance of contamination one can pour them in a microbiology hood.
We typically use 100x20mm (diameter x height) petri dishes, pouring 18 mL in each. 

7. Put the bottle on a stirrer, and wait until it cools down to around 60°C. At this temperature, the bottle should feel very hot to the touch, but one can hold it for a while without feeling too uncomfortable. The agar should still be liquid.
14. We store our NGM plates at room temperature for 3-4 days. We then flip them to avoid condensation in the lid, and store them at 4°C with a cover on each stack to limit evaporation.
TITLE: 0.05 M Glycine
AUTHORS: Jacquelina.Woods  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation. 

[STEPS]
1. Mix 3.75 g  in deionized or ultrapure water to 1 L

2. Adjust pH 9.5

3. Autoclave 121 °C15 min

4. Store at 2-8 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In order to determine the consequence of <em>ILDR1</em> reported mutations on splicing and translation initiation, we used Human Splicing Finder (version 3.0) (http://www.umd.be/HSF3/) and ORF finder (http://www.geneinfinity.org/sms/sms_orffinder.html) respectively.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<strong>Purpose:</strong> This protocol describes how to adsorb viruses onto TEM (transmission electron microscopy) grids. The sample is allowed to sit on a hydrophilic grid and viruses adsorb onto the surface of the grid. This technique is generally used for viral lysates with high concentrations of viruses. For natural samples, use the protocol &ldquo;<a href="https://www.protocols.io/view/Quantitatively-Depositing-Viruses-onto-TEM-Grids-cwzxf5" target="_blank">Quantitatively Depositing Viruses onto TEM Grids using an Airfuge</a>&rdquo;.<br /><br />Note: If purification of the viruses is necessary, refer to Ackermann and Heldal (2010) for various options.

[GUIDELINES]
<strong>Materials required:</strong> <br />- glow discharge apparatus<br /> - EM grid-grade tweezers (2 or more pairs) <br />- EM grids (formvar coated 200 mesh copper)<br /><br /><strong>Note:</strong> If purification of the viruses is necessary, refer to Ackermann and Heldal (2010) for various options.

[STEPS]
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TITLE: Measuring tension and calcium imaging within cell subtypes of the colon in response to vagus nerve stimulation
AUTHORS: Dante Heredia, Thomas Gould

[DESCRIPTION]
Protocol for measuring tension and activity in distinct cell types of the colon in response to stimulation of the vagus nerve in an ex vivo preparation lacking the afferent brainstem targets of the vagus nerve.

[GUIDELINES]
Nothing more than in steps.

[STEPS]
1. The entire gut, including esophagus with vagus nerve trunks attached, and stomach and intestines and mesentery, is dissected and placed in a dish with oxygenated Krebs-Ringers solution.  The small intestine, as well as pancreas and spleen, are removed carefully.  The colon is pinned and gently flushed of its contents.  The right vagus nerve trunk is gently dissected away from the esophagus.  The tissue is perfused with 35-degree, oxygenated Krebs-Ringers for the duration of the experiment. 

2. For tension, the colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon.  An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening. Suture silk is used to connect three force transducers (model TST125C; Biopac Systems, Santa Barbara, CA) to the proximal, transverse and distal segments of the colon. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems). A suction electrode connected to a 10ml syringe and tubing is then placed over the right vagus nerve trunk and upward pressure is applied to suck in the nerve. Stimulation is then performed with a variety of pulse duration durations and frequencies.
3. For calcium imaging, either Kit-GCaMP6f, ChAT-GCaMP6f, or nNOS-GCaM6f mice are used. In this dissection, the middle colon only is slit from the ventral midline, and pinned with gentle tension.  This is the area of the colon that is imaged. A suction electrode connected to a 10ml syringe and tubing is then placed over the right vagus nerve trunk and upward pressure is applied to suck in the nerve. Stimulation is then performed with a variety of pulse duration durations and frequencies, and calcium responses are imaged.
TITLE: DNA Cloning (Gibson Assembly, Transformation, Plating and Incubation)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM Team followed this protocol to construct the plasmid of interest or to clone the plasmid of interest.

[STEPS]
SECTION: Gibson Assembly
1. Calculate the volumes of respective DNA fragments to assemble based on their length and concentration. (The maximum final volume of the mixed fragments is 5 µL for each reaction.) 

SECTION: Gibson Assembly
2. Add the DNA fragments into a PCR tube according to the volumes obtained from the calculation.

SECTION: Gibson Assembly
3. Add 5 µL of NEBuilder HiFi DNA Assembly Master Mix into the same PCR tube.

SECTION: Plating and Incubation
19. Prepare an LB agar plate with the correct antibiotics.
SECTION: Plating and Incubation
20. Remove 950μL of the LB solution from the Eppendorf tube that contains the cell pellet, leaving about 100μL in the Eppendorf tube.
SECTION: Plating and Incubation
21. Resuspend the cells by pipetting the solution.
SECTION: Gibson Assembly
4. Vortex to mix the solution and centrifuge the PCR tube to spin down the remaining solution on the wall.
SECTION: Gibson Assembly
5. Incubate the PCR tube at 50°C for 60 min.

SECTION: Transformation
8. Take an Eppendorf tube that contains   from the -80°C fridge.

SECTION: Transformation
9. Immediately place the Eppendorf tube with competent cells into the ice box for 5 min.

SECTION: Transformation
10. Add 20 µL  of the Gibson Assembly product or 1 µL of pure DNA plasmid into the Eppendorf tube containing the competent cells.

SECTION: Transformation
11. Tap the bottom of the Eppendorf tube to mix the solution.
SECTION: Transformation
12. Leave the Eppendorf tube in ice for 10 min.

SECTION: Transformation
13. Place the Eppendoft tube into a foam floating.
SECTION: Transformation
14. Place them into the water bath for 45 s at 42°C for heat shock.

SECTION: Transformation
15. Place the Eppendorf tube into the ice immediately.
SECTION: Transformation
16. Add 1 mL of the LB  media into the Eppendorf tube.

SECTION: Transformation
17. Place the Eppendoft tube into the incubator at 37°C for 60 min for recovery.

SECTION: Transformation
18. Centrifuge the Eppendorf tube to form a cell pellet (no specific speed and time).
SECTION: Transformation
6. Switch on the water bath and set the temperature to 42°C.
SECTION: Transformation
7. Prepare a box of ice.
SECTION: Plating and Incubation
22. Spread the cells onto the agar with the L-spreader. 
SECTION: Plating and Incubation
23. Place the petri dish in the incubator at 37°C for 60 min to allow the colonies to grow.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is suitable for both endogeneous and overexpressed protein immunoprecipitations.

[STEPS]
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TITLE: Visium CytAssist FFPE -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The Visium CytAssist Spatial Gene Expression for FFPE assay is designed to analyze mRNA in
tissue sections derived from formalin fixed & paraffin embedded (FFPE) tissue samples. The Visium
CytAssist instrument requires a glass slide with intact tissue sections as input. 

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the University of Minnesota Imaging Center and sequenced by the University of Minnesota Genomics Center.



[STEPS]
SECTION: Deparaffinization, H&amp;E Staining, Imaging &amp; Decrosslinking
1.   

SECTION: Library Preparation &amp; Sequencing
2.   

 


SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq software version 2.20.0
TITLE: Dilutions and Pipetting Practice (Instructor Protocol)
AUTHORS: Brian P Teague

[DESCRIPTION]
This is the instructor protocol for
 
It contains setup instructions and instructor tips for running the lab.

[STEPS]
SECTION: Lab Setup
1. In the conical tubes, add 500 µl of food coloring to 9.5 ml of tap water to make the "10X stocks." Make a stock for each color.
SECTION: Lab Setup
2. Transfer 500 µLof each stock into a microcentrifuge tubes. Label them appropriately.

SECTION: Instructor Tips &amp; Common Student Errors
4. Instructor Tips
I often start the class by having students grab a P-1000 or P-200, setting them to 500 and 200 µl respectively, and together slowly pressing down to find the first stop, then the second. We transfer a bit of water together, then I leave them to their own.
Students often recognize c1 * v1 = c2 * v2 but have never actually used it (or have forgotten how to use it) to actually make dilutions. The original protocol, from which this was adapted, has quite explicit instructions. I usually allow students to struggle, ask them to consult their colleagues, etc, and then when a group is properly stuck will help them through it.   
Stand by the standards and compare students' results with them. Students often don't have a good idea of how close is "close enough." Both the volumes and colors should be precisely the same!
Comparing students' samples to standards is easier if you set the standards on a piece of white paper.
If a student's sample is substantially different, ask them to go back and try it again. 

 

If, after a second attempt, a student sample isn't correct, follow them back to their bench and observe their technique closely. 

 

SECTION: Lab Setup
3. Follow the student protocol to create standards.
SECTION: Instructor Tips &amp; Common Student Errors
5. Common student errors
Pressing the plunger all the way down (instead of to the first top) when aspirating, and thus aspirating too much liquid.

 


Not watching their pipetting, but doing so blind (especially for the 1/10th scale).
Making their "mixed" samples from the 10X concentrate instead of the 1X working stocks.
Forgetting that to make a dilution, they need to add both the concentrate and a diluent (in this case, water).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The following protocol pools miniaturized PCR replicates from (2) different 384-well plates into (1) final 384-well plate using the epMotion 5075. The miniaturized PCR reaction was done in triplicate across (3) 384-well plates and the third PCR plate will be the final storage plate for PCR products. 

[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Prepare PCR plates
?. 
SECTION: Setup epMotion automation platform
?. 
SECTION: Execute automated protocol
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TITLE: DNA Extraction
AUTHORS: Stefania Konstantinidi, Hana Samet

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The goal is to extract DNA from plant leaves using a microneedle patch.</div></div>

[STEPS]
?. [Application of microneedle patch]
Place the microneedle patch between the base and the midrib of the leaf and apply pressure for a few seconds.
?. [Application of microneedle patch]
Carefully remove the microneedle and put is in a petri dish.
?. [Application of microneedle patch]
Add 100uL of EB buffer on the microneedle patch using a pipet, and then put the EB buffer and DNA in a tube.
?. [Application of microneedle patch]
The DNA is ready for amplification !
TITLE: Nuclei Isolation from Human Cartilage for 10x Multiome
AUTHORS: Takuya Sakamoto, Erpei Wang, Merissa Olmer, Martin Lotz

[DESCRIPTION]
This protocol describes isolation of nuclei from fresh-frozen human knee cartilage for use in Omics analyses, including RNA-sequencing of ATAC-sequencing. Tissue dissociation was the most critical step in optimization which was monitored by nuclei yield, integrity and purity. Methods for quality control are also described.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>In the study we have used a standard Thermo Scientific TaqMan protocol which can be found under this link:</p>
</div>
<div> </div>
<div>http://tools.thermofisher.com/content/sfs/brochures/taqman-assay-genetic-variation-research-br.pdf</div>

[GUIDELINES]
<div>
<p>In the study we have used a standard Thermo Scientific TaqMan protocol which can be found under this link:</p>
</div>
<div> </div>
<div>http://tools.thermofisher.com/content/sfs/brochures/taqman-assay-genetic-variation-research-br.pdf</div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
K-mer-based approaches to determining sequence similiarity can be faster than traditional use of BLAST.  This application uses <a href="http://www.genome.umd.edu/jellyfish.html" target="_blank">Jellyfish</a> to index host FASTA files, then uses the mode value (default 2) of the number of matching k-mers (default k=20) from the input sequence to determine whether the sequence is similar enough to the host sequence to be rejected.  The output of the app is:<br /><br />
<ul>
<li>"screened" directory containing the sequences from each file that were found to be dissimlar to the host</li>
<li>"rejected" directory containing the sequence from each file that were too similar to the host</li>
<li>"jf" directory containing Jellyfish indexes of each "host" file (useful for later runs with other files to skip recreating)</li>
<li>"kmer" directory containing k-mers of query sequences and ".loc" file showing the number of ".kmer" lines associated to each sequence</li>
</ul>
Code is freely available at <a href="https://github.com/hurwitzlab/docker-screen-host" target="_blank">Github</a>.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Este protocolo describe como aislar bacterias mótiles del género P</span><em><span style="font-weight: 400;">aenibacillus</span></em><span style="font-weight: 400;"> a partir de muestras ambientales. El protocolo no involucra material de laboratorio especializado.</span></p>
<p><span style="font-weight: 400;">Parte del proyecto Comunicaciones Especulativas: </span></p>
<p><span style="font-weight: 400;">datos en https://osf.io/c542q/wiki/home/</span></p>
<p><span style="font-weight: 400;">codigo en https://github.com/interspecifics/ComunicacionesEspeculativas</span></p>
<p><span style="font-weight: 400;">info en http://interspecifics.cc/comunicacionesespeculativas/</span></p>

[BEFORE_START]
<p>Antes de realizar la recolección se deben tener preparados los contenedos limpios o placas de LB 1.5% agar (en caso de plaqueo directo).Además, se debe tener preparado y autoclavado el glicerol 50% antes de preprar los stock criogénicos.</p>

[GUIDELINES]
<p><span style="font-weight: 400;"><em>Paenibacillus sp.</em> es una bacteria mótil presente comunmente en el suelo. De esta forma puede ser colectada a partir de muestras de tierra, suelo, polvo, pelos de animales, etc.</span></p>
<p><span style="font-weight: 400;">Su identificación puede ser realizada simplemente por inspección visual, por lo que no es necesario utilizar antibióticos ni tinciones especializadas.</span></p>

[STEPS]
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