eamag/protocols-with-steps huggingface dataset:
TITLE: Leucine auxotrophy test of Fusarium graminearum strains
AUTHORS: martin.urban

[DESCRIPTION]
This protocol outlines the methodology for preparing Leucine dropout medium, essential for assessing Leucine auxotrophy in Fusarium graminearum strains. The procedure involves the step-by-step preparation of the medium to selectively evaluate the growth characteristics of these strains.  Understanding the Leucine auxotrophy status in Fusarium graminearum strains holds significance in elucidating their metabolic pathways or assign phenotypes to mutant strains.

[STEPS]
1. Testing Fusarium graminearum strains for Leucine Auxotrophy 

Media and supplements:

 Biotin (Supplier: Fluka/Merck) 
     Biotin is essential for robust growth, particularly beneficial for vivid photography on minimal 
     medium.

    Procedure:
Dissolve in water to achieve a 0.05% solution.
Add 20 µl of the 0.05% Biotin stock to 400 ml agar.
     
Biotin, while not strictly necessary for growth. leads to more vigorous growth,  facilitating better photography of the mycelic growth. There might be a role for biotin in the initial steps of glucose metabolism  (see references: STRIGINI, P., MORPURGO, G. (1961) and Wu, P.-C., et al. (2020)).

2. Yeast Nitrogen Base (Formedium, CYN0202) 
     This complete medium is used for routine growth of all strains before the auxotrophy test.

3. Synthetic complete (Kaiser) Drop-out: -LEU   (supplier: Formedium, Ref no: DSCK052)
     This is the test medium.

4. L-Leucine (supplier Formedium, Ref no: DOC0155)

 Procedure:
Dissolve 1.312 g  L-Leucine in 1 M HCL to produce a 1 M stock solution of L-Leucine.

2. Preparation for 400 ml YNB agar and the Kaiser Drop-out agar  with and without Leucine

Medium Composition:
Yeast Nitrogen Base (YNB): 1.76 g      (contains all essential aminoacids)
Kaiser Drop-out: -LEU: 2.76 g             (contains all aminoacids but not Leucine)
2% agar

 Procedure:
 Weigh YNB and Kaiser Drop-out  medium into two 500 ml Schott glass media bottles,
        and add 8 g agar.
Add 400 ml deionised water and autoclave for 15 minutes.
After autoclaving, cool agar to 50 C.
Pour YNB medium into agar plates
For the Kaiser Drop-out medium, add 20 ul 0.05% Biotin stock to 400 ml agar and mix.
Divide the 400 ml agar into two 200 ml aliquots using sterile Schott glas bottles.
Pour one aliquot into agar plates to produce the Leucine drop-out medium.
For the remaining 200 ml aliquot, add 400 ul  1M L-Leucine to 200 ml agar to produce a medium containing  2 mM Leucine.


3. Growth test of Fusarium graminearum strains
F. graminearum strains are first grown on YNB agar. Small agar plugs (3 x 3 mm2) taken from the actively growing end of the colony are then transferred on agar plates with Kaiser Drop-out medium with and without added Leucine. Plates are incubated at 25 C and growth is assessed after 5 days.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Thes protocols were original created by <a href="&quot;http://www.whoi.edu/sbl/liteSite.do?litesite" target="_blank">Krista Longnecker</a> and <a href="http://jamesrco.github.io/" target="_blank">Jamie Collins</a> for creating lipsomes to be used in lipid photo-oxidation experiments. The results of these experiments are detailed in Chapter 4 of:</p>
<p> </p>
<p>Collins, J. R. 2017. The remineralization of marine organic matter by diverse biological and abiotic processes. Ph.D. thesis. Cambridge, Massachusetts: Massachusetts Institute of Technology, 300 pp; doi:<a href="http://dx.doi.org/10.1575/1912/8721" target="_blank">10.1575/1912/8721</a></p>
<p> </p>
<p>A manuscript of the chapter is forthcoming.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Preparation-of-Denaturing-Agarose-Gel-for-RNA-Anal-es4begw" target="_blank">Preparation of Denaturing Agarose Gel for RNA Analysis</a>.

[STEPS]
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TITLE: Effect of postoperative coffee consumption on postoperative ileus after abdominal surgery: An updated systematic review and meta-analysis (protocol)
AUTHORS: Jun Watanabe, Atsushi Miki, Kazuhiko Kotani, Naohiro Sata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Introduction:</span><span> Coffee, a popular and cheap beverage worldwide, may have an important effect on postoperative ileus (POI). However, previous systematic reviews have not clarified whether the effect is due to caffeine or coffee itself, or shortening hospital stay. We will aim to assess the effect of postoperative coffee consumption on POI.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Methods:</span><span> Studies evaluating the effect of postoperative coffee consumption will be searched using the electronic databases and the trial registries. Meta-analyses will be performed using random-effects models. The Grading of Recommendations, Assessment, Development, and Evaluation approach will be used to assess the certainty of evidence.</span></div></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In this protocol, the incubation of <em>Synechocystis</em> sp. PCC 6803 colonies on agarplates and further incubation in flasks for further plate reader measurements for fluorescence detection are described. The protocol was handed over by Anna Behle MSc.</p>

[BEFORE_START]
<p>Prepare BG-11 media-based agar plates.You can use the <a href="https://www.protocols.io/view/recipe-for-standard-bg-11-media-gtmbwk6" target="_blank">recipe for standard BG-11 media</a>, which includes the recipe for BG-11 media based agar plates.</p>
<p>Order/borrow <strong>agar plates, fresh cultures or cryo cultures</strong> with your <strong>desired colonies</strong>. If your agar plates are contaminated, make sure that you only pick <strong>uncontaminated</strong> colonies.</p>

[STEPS]
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TITLE: Coating plates with Matrigel for ES/iPS culture
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about coating plates with matrigel.</div></div>

[STEPS]
?. Chill .
[PBS]
on ice
Skip this step if you have PBS in the fridge already.
?. Take culture dishes you want to coat out of their package and place them inside a tissue culture hood.
?. Thaw desired number of Matrigel aliquots either  or at .
on ice
0 Room temperature
Keep in mind that matrigel will solidify at room temperature. So if you thaw matrigel at room temp, you want to make sure to proceed to the next step before the gel is warmed up to room temperature. You can proceed to the next step even if there is still a little some frozen matrigel in the tube. PBS should thaw them quite quickly.
?. Resuspend matrigel with chilled PBS. You want to use  per .
[PBS]
[matrigel]
?. Make sure the matrigel is well mixed in the PBS. Then aliquot proper volume into each culture dish. You will need  per 10 cm dish,  per well of a 6 well plate,  per well of a 12 well plate.Swirl plate to coat all the surface.
6 mL
1 mL
0.5 mL
?. These plates are ready to use after  at . Depending on the storage space situation. You can keep these plates in a  tissue culture incubator, somewhere clean at  or in the fridge. As long as they are kept sterile and the solution is not dried out, they are good for a couple months. Dried out plates have to be either discarded or re-coated.
0 Room temperature
37 °C
37 Room temperature
?. After , add  on top to avoid the wells from drying out.
[fresh 1xPBS ]
TITLE: Intraperitoneal Injection in an Adult Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the general procedure used for intraperitoneal injection in adult mice.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the protocol for dephosphorylation of 5'-ends of DNA using AnP (Antarctic Phosphatase - M0289).

[GUIDELINES]
Dephosphorylation of 5' -ends of DNA in Restriction Enzyme Reaction
The phosphate can be added directly into the digestion reaction during or after DNA digestion
Antarctic Phosphatase is active in all NEB restriction enzyme buffers only when supplemented with Antarctic Phosphatase Reaction Buffer, which provides Zn2+ required for enzyme activity
The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
If restriction enzyme cannot be heat inactivated, DNA purification is required before ligation

[STEPS]
1. Prepare a 20 µL reaction as follows:
 

2. Incubate at 37 °C for 30 min.
3. Stop reaction by heat-inactivation at 80 °C for 2 min.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Preparation of the trace metal mixture for addition to seawater for the cultivation of marine cyanobacteria, <em>Prochlorococcus</em> and <em>Synechococcus</em>

[BEFORE_START]
Prepare the six trace metal stock solutions as described in the Primary Trace Metal Stocks protocol:<br />
<ul>
<li>0.08M ZnSO<sub>4</sub>·7H<sub>2</sub>O ((2.30 g/100 mL)</li>
<li>0.05M CoCl<sub>2</sub>·6H<sub>2</sub>O (1.19 g/100 mL)</li>
<li>0.90M MnCl<sub>2</sub>·4H<sub>2</sub>O (17.81 g/100 mL)</li>
<li>0.03M Na<sub>2</sub>MoO4·2H<sub>2</sub>O (0.726 g/100m mL)</li>
<li>0.10M Na<sub>2</sub>SeO<sub>3</sub> (1.73 g/100 mL)</li>
<li>0.10M NiCl<sub>2</sub>·6H<sub>2</sub>O (2.38 g/100 mL)</li>
</ul>

[GUIDELINES]
All chemicals used for cultivating cyanobacteria should be of the highest quality.<br />All re-useable glassware and plastic should be acid-washed and autoclaved.<br />Use only plastic or teflon spatulas or dust-free weigh paper for weighing out chemicals.

[STEPS]
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TITLE: Microglia FACS staining after isolation (from UCSD)
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details about microglia FACS staining after isolation (from UCSD).

[GUIDELINES]
Gating Notes
live vs dead
FSC vs SSC
singles vs. doublets.
CD11B (high) vs. CD45 (low).
CX3CR1 (optional)  gate on positive cells.

Misc. Notes
**don’t need to compensate with only 2-3 colors.
CD45, CD11B, and CX3CR1 are all surface markers.
Usually get 20-50% live cells after staining.
Better to do staining right after microglia prep rather than waiting O/N for staining.

Processing Notes
Can do RNA seq or Atac Seq
RNA Seq
Spin down sample in Eppendorf (not in facs tubes
Remove supernatant
Add 150 µL of trizol → resuspend 
Store in -80 °C (can keep stored for months).
ATAC Seq
Do transposase reaction and then freeze for processing.


[STEPS]
SECTION: Protocol
1. Resuspend cells staining buffer (300 µL of HBSS+EDTA+BSA).
Resuspend in a 15-ml falcon tube.
SECTION: Protocol
2. Add Fc Block (~1:100 dilution) and incubate for 15 min at 4 °C or in the fridge.
Add this to cells resuspended in the HBSS.
SECTION: Protocol
3. Take ~5% of cells for unstained control, put on ice. 
 


SECTION: Protocol
4. Add antibodies (CD11b, CD45, 1:100 dilution) for 20-30 min at 4 °C.
SECTION: Protocol
5. Add DAPI, 45 s, then dilute with HBSS+BSA+EDTA.
SECTION: Protocol
6. Put 70-μm filter on top of a FACS test tube and filter resuspended pellet into the tube.
 


SECTION: Protocol
7. Centrifuge for 400 x g, 10 min.
SECTION: Protocol
8. Vacuum out supernatant.
Remove most of supernatant; not all.


SECTION: Protocol
9. Resuspend pellet in 500 µL to 1000 µL staining buffer.
 

SECTION: Protocol
4.1. Add directly to cells in the FC block.
SECTION: Protocol
4.2. Mix by tapping tube; do not vortex.
 

SECTION: Protocol
5.1. Add 1:1000 dilution.
SECTION: Protocol
5.2. Add to unstained cells.
SECTION: Protocol
6.1. Push filter hard onto tube.
SECTION: Protocol
7.1. Prepare collection tubes during spin time.
Coat FACS tubes with 1 mL of staining buffer to prevent cells from sticking to walls of tubes.
Invert tube several times.
TITLE: Autofluorescence Microscopy QC for Multimodal Molecular Imaging 
AUTHORS: Katerina V Djambazova, Nathan Heath Patterson, Lukasz Migas, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins

[DESCRIPTION]
Autofluorescence QC is performed prior to collecting on-tissue AF. Fluorescence intensity can be tracked for each fluorescence channel (DAPI, EGFP, and DsRed), allowing the microscope performance and the LED light source to be benchmarked and tracked over time.

[STEPS]
SECTION: Autofluorescence Microscopy QC
1. Fluorescence QC is collected prior to any on-tissue AF collection.

Use fluorescence calibration slides - StarLight Calibration Slides - Bang Laboratories 
DAPI (excitation-360nm, emission-450nm), Dragon green (excitation-480nm, emission-520nm), and Envy green (excitation-525nm, emission-565nm) 
SECTION: Autofluorescence Microscopy QC
2. Load calibration slides into Zeiss AxioScan.Z1 slide scanner
SECTION: Autofluorescence Microscopy QC
3. Select the same ~ 25 x 25 mm square region in each calibration slide
 

SECTION: Autofluorescence Microscopy QC
4. Acquire fluorescence microscopy data for each calibration slide.
 

SECTION: Autofluorescence Microscopy QC
5. Data Analysis:
Once data are collected, background pixels are subtracted, and the average intensity of each channel is calculated and monitored over time.
TITLE: Electroporation Protocol
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol may be used with electrocompetent cells prepared by you according to </span><a href="https://www.protocols.io/view/Making-your-own-electrocompetent-cells-imsv6m" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">this protocol.</span></a></div></div>

[STEPS]
?. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF
?. Place recovery SOC in 37°C water bath
?. Pre-warm LB-antibiotic plates at 37°C
?. Thaw cells on ice for 10 min or use freshly made cells
?. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice
?. Flick the tube containing cells a few times to mix and add 25 µl of competent cells to the microcentrifuge tube
25 µl
?. Add 1 µl of a 100 pg/µl to 1 ng/µl DNA solution (in DI water) to the cells in the microcentrifuge tube
1 µl
?. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (you don’t hold the button down)
?. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a microcentrifuge tube, 5 ml culture tube, or 15 ml centrifuge tube.
975 µl
?. Place in the shaker/incubator at 37°C incubator for 1 h
?. Make appropriate dilutions
When using 100 pg - 1 ng of DNA, make three dilutions:Dilute 1 µl of cells into 990 µl SOC and plate 100 µl. (10000-fold dilution)Dilute 10 µl of cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)Dilute 100 µl of cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
?. Incubate overnight at 37°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a DNA extraction protocol from sea cucumber (Apostichopus japonicus) body-wall tissue. It accompanies the following <em>GigaScience</em> publication:</p>
<p> </p>
<p>Jihoon Jo, et al.  (2016): Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants. <em>GigaScience</em>...</p>

[BEFORE_START]
<p>Pre-heat the waterbath or hot-plate to lysis buffer incubation, and pre-heat the CTAB lysis buffer at 65℃.</p>

[STEPS]
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TITLE: Microtome Dissection of Beetles
AUTHORS: Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how visualize internal structures, from mandibular mycangia, to beetle heads or bodies.</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: Surface protein biotinylation
AUTHORS: Daehun Park, Pietro De Camilli

[DESCRIPTION]
This protocol describes surface protein labeling with biotin using EZ-Link Sulfo-NHSLC-Biotin. This chemical reacts with primary amines such as lysine but does not  permeate cell membranes because of the charge. Thus, it only biotinylates surface  proteins.

[STEPS]
SECTION: Surface protein biotinylation
1. Wash cells with ice-cold PBS.
 
SECTION: Surface protein biotinylation
3. Discard biotin containing medium. Quench and remove unbound biotin using 50 millimolar (mM) glycine in ice-cold PBS for 10 min at4 °C.

SECTION: Surface protein biotinylation
4. Wash 2-3 times with ice-cold PBS. 
SECTION: Surface protein biotinylation
6. Collect supernatants and discard the pellets.
SECTION: Surface protein biotinylation
7. Measure the protein concentrations using BCA Protein Assay Kit.
SECTION: Surface protein biotinylation
8. Incubate the same amount of lysates (500 µg to 1000 µg) with streptavidin or NeutraAvidin beads for 120 min to 120 min at4 °C to pull-down the biotinylated proteins.

SECTION: Surface protein biotinylation
9. Wash the beads with lysis buffer by cycles of suspension.

SECTION: Surface protein biotinylation
11. Run eluate samples on a SDS polyacrylamide gel and perform western blotting to visualize labeling (and thus evidence of surface expression) of the protein of interest. Total cell lysates can be used to determine the expression level.
SECTION: Surface protein biotinylation
2. Incubate cells with EZ-Link Sulfo-NHS-Biotin for  30 min at 4 °C to label surface proteins.
 

SECTION: Surface protein biotinylation
1.1. Wash cells with ice-cold PBS. (1/3)
SECTION: Surface protein biotinylation
1.2. Wash cells with ice-cold PBS. (2/3)
SECTION: Surface protein biotinylation
1.3. Wash cells with ice-cold PBS. (3/3)
SECTION: Surface protein biotinylation
5. Lysis cells with 1% triton X-100 lysis buffer and centrifuge the samples at 14000 x g, 20 min, 4 °C.

SECTION: Surface protein biotinylation
4.1. Wash with ice-cold PBS. (1/3)
SECTION: Surface protein biotinylation
4.2. Wash with ice-cold PBS. (2/3)
SECTION: Surface protein biotinylation
4.3. Wash with ice-cold PBS. (3/3)
SECTION: Surface protein biotinylation
10. Centrifugation and elute proteins from the packed beads by adding an equal volume of 2x sample buffer and boiling for 5 min 95 °C.
SECTION: Surface protein biotinylation
9.1. Wash the beads with lysis buffer by cycles of suspension. (1/3)

SECTION: Surface protein biotinylation
9.2. Wash the beads with lysis buffer by cycles of suspension. (2/3)
SECTION: Surface protein biotinylation
9.3. Wash the beads with lysis buffer by cycles of suspension. (3/3)
TITLE: MIDAS 2 Protocol
AUTHORS: miriam.goldman  , chunyu.zhao  

[DESCRIPTION]
The Metagenomic Intra-Species Diversity Analysis System 2 (MIDAS2) is a scalable pipeline that identifies single nucleotide variants (SNVs) and gene copy number variants (CNVs) in metagenomes using comprehensive reference databases built from public microbial genome collections (“metagenotyping”). MIDAS2 is the first metagenotyping tool with functionality to control metagenomic read mapping filters and to customize the reference database to the microbial community, features that improve the precision and recall of detected variants. Here we present four basic protocols for the most common use cases of MIDAS2, along with two supporting protocols for installation and advanced use. All the steps of metagenotyping, from raw sequencing reads to population genetic analysis, are demonstrated with example data in two downloadable sequencing libraries of single-end metagenomic reads representing a mixture of multiple bacterial species. This set of protocols empowers users to accurately genotype hundreds of species in thousands of samples, providing rich genetic data for studying the evolution and strain-level ecology of microbial communities.

[STEPS]
TITLE: High-throughput phenotyping of green canopy area during nematode infection
AUTHORS: Jaap-Jan Willig, Devon Sonneveld, Joris J.M. van Steenbrugge, Laurens Deurhof, Casper C. van Schaik, Misghina G. Teklu, Aska Goverse, Jose L. Lozano Torres, Geert Smant, Mark G. Sterken

[DESCRIPTION]
Nematode migration, feeding site formation, withdrawal of plant assimilates and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognised as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana, since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode-plant interactions. We established through imaging of tolerance-related parameters that green canopy area was an accessible and robust measure for assessing damage as the consequence of cyst nematode infection. Subsequently, we developed a high-throughput phenotyping platform to non-destructively measure the green canopy area growth of 960 A. thaliana plants simultaneously.

[STEPS]
1. Fill 200mL pots with silversand.
2. Place the pots in the steel frames.
None. 
2.1. Once all the pots are filled with silversand, cover them with the black nonreflective foamed PVC coversheet. 
4. Sow 2 to 5 Arabidopsis seeds per pot using a toothpick.
3. Prior to sowing, silver sand was watered with Hyponex for five minutes.
5. Seven days after sowing, remove redundant seedlings were using a tweezer.
5.1. At eight days after sowing, water the plants with Hyponex for five minutes.
6. Inoculate the seedlings with nematode at nine days after sowing
7. After inoculation, start the camera system to take pictures of the Arabidopsis seedlings (15 pictures per day with an hour interval for a period 23 days).
8. Finally, the surface area of the rosette was determined using a custom-written ImageJ macro (ImageJ 1.51f; Java 1.8.0_321 [32-bit]) and Java was used to make GIFs.