eamag/protocols-with-steps huggingface dataset:
TITLE: Preliminary qualitative phytochemical analysis
AUTHORS: Matin Mahmood, Abdulkareem Hameed Abd, Enas Jawad Kadhim

[DESCRIPTION]
Plant extraction is a process that aims to extract certain components present in plants. It is a solid/liquid separation operation: a solid object (the plant) is placed in contact with a fluid (the solvent). The plant components of interest are then solubilized and contained within the solventChemical tests were carried out using the ethanolic extracts from plants and different fractions, using standard procedures to identify the active constituents

[STEPS]
SECTION: Test for alkaloids
1. Ten (2ml) of alcoholic extract and fractions were stirred with 5 ml of 1% HCL on a steam bath. 
SECTION: Test for alkaloids
2. Mayer’s (1.35gm mercuric chloride in 60ml water + 5gm potassium iodide in 10ml water )and Wagner’s reagents (1.27g of iodine and 2g of potassium iodide in 100ml of water) were added, white and reddish brown color precipitate respectively, were taken as evidence for the presence of alkaloids
SECTION: Test for flavonoids
3. Two tests used for detection of flavonoids 
SECTION: Test for flavonoids
3.1. Lead acetate test: to 2ml of alcoholic extract and fractions, 1ml of 10% lead acetate solution was added. The formation of a yellowish- white precipitate was taken as a positive test for flavonoids
SECTION: Test for flavonoids
3.2. NaOH test: 2ml of the extract and fractions were treated with aqueous NaOH and HCl, and looking for the formation of a yellow orange color
SECTION: Tests for steroids
4. Two tests used for identification of steroids 
SECTION: Tests for steroids
4.1. Liebermann-Burchard test: 2ml of the extract and fractions were treated with chloroform, acetic anhydride and drops of sulphuric acid was added. The formation of dark pink or red color indicates the presence of steroids
SECTION: Tests for steroids
4.2. H2SO4 test: The development of a greenish color was considered as indication for the presence of steroids, when 2 ml of the extract and fractions were treated with sulphuric and acetic acids.
SECTION: Test for terpenoids
5. Two ml of the organic extract and fractions were dissolved in 2 ml of chloroform 
SECTION: Test for terpenoids
6. Evaporated to dryness
SECTION: Test for terpenoids
7. 2 ml of concentrated sulphuric acid was then added and heated for about 2 min. A grayish color was considered an indication for the presence of terpenoids.
TITLE: KAPP-Sen TMC: 10x Visium CytAssist FFPE
AUTHORS: William F. Flynn, Paul Gabriel Jr., Emily C. Soja, Grace K. Frohock, Jessica D. S. Grassmann, Allison D. McNeilly, Sandra L. Daigle, Greggory Perry, Anahita Amiri, Shannon Bessonett, Diane Luo, Marie Jacques Martine Seignon, Elise T. Courtois, Paul Robson

[DESCRIPTION]
The Visium CytAssist Spatial Gene Expression workflow assays RNA levels in intact formalin-fixed paraffin embedded (FFPE) tissue sections. It uses probes targeting the whole transcriptome. Each Visium CytAssist Spatial Gene Expression Slide contains Capture Areas with barcoded spots that include oligonucleotides required to capture gene expression probes.

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the Single Cell Biology Core at The Jackson Laboratory for Genomic Medicine.

[STEPS]
SECTION: Deparaffinization, H&amp;E, Decrosslinking
1. Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, H&E Staining, Imaging & Decrosslinking: CG000520 Rev B

  

SECTION: Library Preparation
2. Visium CytAssist Spatial Gene Expression Reagent Kits User Guide: CG000495 Rev E

 

Library concentration was quantified using a Tapestation High Sensitivity DNA ScreenTape (Agilent) and fluorometry (Thermofisher Qubit) and verified via KAPA qPCR.
SECTION: Library Sequencing
3. Libraries were pooled for sequencing on an Illumina NovaSeq 6000 200 cycle flow cell using a 28-10-10-90 read configuration, targeting 100,000 read pairs per spot covered by tissue.

  

SECTION: FASTQ Generation
4. Illumina base call files for all libraries were converted to FASTQs using bcl2fastq v2.20.0.422 (Illumina).

https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html
TITLE: Protocols for "Shotgun Metagenomics of 361 elderly women reveals gut microbiome change in bone mass loss"
AUTHORS: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bone mass loss contributes to the risk of bone fracture in the elderly. Many factors including age, obesity, estrogen and diet, are associated with bone mass loss. Mice studies suggested that the gut microbiome might affect the bone mass by regulating the immune system, however there has been little evidence from human studies. </div><div class = "text-block">Bone loss increases after menopause. Therefore, we have recruited 361 Chinese post-menopausal women to collect their fecal samples and metadata to conduct metagenome-wide association study (MWAS) to investigate the influence of the gut microbiome on bone health. Gut microbiome sequencing data were produced using BGISEQ500 sequencing, Bone mineral density (BMD) was calculated using Hologic dual energy X-ray machine, body mass index (BMI) and age were also recorded.This collected data allows exploration of the gut microbial diversity and their links to bone mass loss, as well as microbial markers for bone mineral density. In addition, these data are potentially useful in studying the role the gut microbiota might play in bone mass loss and in exploring the bone mass loss process.</div></div>

[STEPS]
TITLE:  Preparation of M9 worm buffer
AUTHORS: Alfonso Pérez Escudero, Alid  Al-Asmar, Gabriel Madirolas

[DESCRIPTION]
Preparation of M9 worm buffer, used to maintain Caenorhabditis elegans in liquid for short periods of time. Protocol taken from the WormBook Methods http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html

[STEPS]
1. Add 3 g of monopotassium phosphate (KH2PO4) in a 1L clean bottle.

2. Add 7.52 g of disodium phosphate dihydrate (Na2HPO4.2H2O).

3. Add 5 g of sodium chloride (NaCl).

4. Add 1 L of milliQ water.

5. Autoclave.
6. Add 1 mL of magnesium sulfate solution. Our protocol for making this solution can be found here:
TITLE: Vandy - Exercise Stress Test
AUTHORS: Louise Lantier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
The exercise stress test is a maximal exercise test used to calculate VO2 max. Mice are run to complete exhaustion as the treadmill speed is increased every three minutes (See table 1). During the test, direct measurements of the oxygen consumption and carbon dioxide output by the mouse are made, which are then used to calculate VO2 max, VCO2, and RER. VO2 max refers to the maximum amount of oxygen that the mouse can utilize during maximal exercise.</div></div>

[STEPS]
?. Mice are familiarized with the treadmill two days prior to the experiment by exercising them for 10 minutes at 10m/min.
?. On the day of the experiment, the mouse is placed in the stopped treadmill for 45 min to acclimatize and the Oxymax system is started. The treadmill is equipped with a shock pad. Shocks are set at 1.5mA, 200ms pulses, 4Hz.
?. After 45 min, basal measurements are made for 15 min in the stopped treadmill.
?. Treadmill is started at 10m/min.
?. After 3 min, speed is increased by 4m/min.
?. Every three minutes thereafter, speed is increased by 4m/min until mouse has reached exhaustion (table 1). Exhaustion is defined as the point at which the mouse remains on the shock pad for 5 continuous seconds.
?. Once exhaustion is reached, the mouse is left 30 min in the stopped treadmill to recover.
TITLE: Protocol for &#34;quantification of neurons expressing alpha-synuclein in the non-human primate brain&#34;
AUTHORS: jlanciego

[DESCRIPTION]
Here we describe a method based on an AI deep learning algorithm (Aiforia) for the purpose of accurately quantifying the number and location of neurons expressing alpha-synuclein throughout pre-defined regions of interest (cortical and sub-cortical locations) in the non-human primate brain.

[STEPS]
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
1. Scan sections to be analyzed in the Aperio CS scanner at a magnification of 20x
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
2. Upload digitalized sections to the Aiforia cloud (www.aiforia.com).
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
3. Create a dedicated project for analysis.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
4. Select up to 3-4 sections for training the model algorithm, these sections showing different morphologies and intensity levels of alpha-synuclein expression.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
5. Train the model algorithm to analyse potential errors.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
6. Activate the annotation assistant tool to retrieve additional beneficial inputs that can be added by the platform itself.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
7. Repeat steps 5 to 7 while increasing the number of annotations and iterations (>1,000) until the model algorithm has learned to properly identify every single phenotype of alpha-synuclein-expressing neurons.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
8. Release the model algorithm.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
9. Use the drawing tool to delineate the boundaries of each selected region of interest where positive neurons need to be quantified in every single section of the project.
SECTION: Aiforia-based quantification of neurons expressing alpha-synuclein
10. Export obtained raw data to an Excel spreadsheet for statistical analysis.
TITLE: Binding of gp37 LTF needle trimers to E. coli
AUTHORS: Mohammad Z. Islam, Andrei Fokine, Marthandan Mahalingam, Zhihong Zhang, Carmela Garcia-Doval, Mark J. van Raaij, Michael G. Rossmann, and Venigalla B. Rao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is based on that described by Blin and Stafford (1976).</p>
<p> </p>
<p>Blin, N., Stafford, D., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Research. 3<strong>,</strong> 2303.</p>
<p> </p>
<p>It uses RNAse and ProteinaseK follwed by phenol/Chlorofrom extraction.  For the highest molecular weight, the DNA is purified by dialysis, avoiding alcohol precipitation.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Cytotoxicity assay using LLC-MK2 cell line
AUTHORS: luanaborba 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Protocol that described the cytotoxicity assay </span></a><span>using a mammalian epithelial cell line (LLC-MK2; ATCC CCL-7).</span></div></div>

[STEPS]
TITLE: Protocol to Capture, Annotate, and Grade produce items using the AI enabled Grader App
AUTHORS: Anjali Sharma

[DESCRIPTION]
This protocol describes steps to capture, annotate, and label produce items as good or bad using the Grader App. The grader app is part of a larger system of systems conceived, designed, and developed by Anjali Sharma at SDG Labs, to reduce food waste using AI and robotics. The grader app is designed to leverage the power of citizen sourcing and AI to create an open image data set of good and bad produce items. The purpose of creating this open image data set is to help in training machine vision algorithms that can accurately differentiate between good and bad produce items. In this protocol I outline the steps following which, any citizen scientist can capture, annotate, and/or label good and bad produce items.

[STEPS]
TITLE: NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II FS DNA Module (NEB # E7810)
AUTHORS: New England   Biolabs, jbonnevie

[DESCRIPTION]
This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina®-compatible library construction.

The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

[BEFORE_START]
Starting Material: 100 pg–500 ng fragmented, end repaired and dA-Tailed DNA generated using the NEBNext Ultra II FS DNA Module (NEB #E7810)
 


[GUIDELINES]
Safe Stop Point: This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol. 

Caution: Signifies a step in the protocol that has two paths leading to the same point. 

Color: A color listed before or after a reagent name indicates the cap color of the reagent to be added.


Adaptor Dilution Guidelines

The appropriate adaptor dilution for your sample input and type may need to be optimized experimentally. The dilutions provided here are a general starting point.

Table 2.1: Adaptor Dilution 

 


[STEPS]
SECTION: DNA Ligation/End Prep
1. Add the following components directly to the FS Reaction Mixture:
 
* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in NEBNext Oligo kit options, which can be found at www.neb.com/oligos
 

 

 

 

SECTION: DNA Ligation/End Prep
2. Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
 

SECTION: DNA Ligation/End Prep
3. Incubate at 20 °C for 15 min in a thermocycler with the heated lid off.
SECTION: DNA Ligation/End Prep
4. Add 3 µl of (red) USER Enzyme to the ligation mixture.
 

 

SECTION: DNA Ligation/End Prep
5. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C. 

SECTION: DNA Ligation/End Prep
6. DNA is now ready for size selection or cleanup.
TITLE: Rapid column Ag test
AUTHORS: Anne Berking

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The "Rapid Ag test based on Alpaca Nanobodies" is a test developed with single domain antibodies. This type of antibody is capable of effectively recognize the Spike protein on the surface of SARS-CoV2 leading a signal that is 10 times stronger than conventional antibodies. </div><div class = "text-block">It takes only 16  minutes from sample to result. The procedures for using this test were designed to ensure an easy use and interpretation, making it a quick and simple test.</div></div>

[STEPS]
?. [Sample Collection]
Add 3 mL of collection buffer to sample collection tube and collect saliva in sample collection tube (3-5mL) wait 5 minutes
?. [Sample Analyse]
Mix binding buffer tube, load 1 drop of binding buffer into Column connected to a vacuum device
?. [Sample Analyse]
Load sample into column, turn on vacuum device, and let sample go through column wait 5 minutes
?. [Sample Analyse]
Add 12mL of washing buffer 1, turn on vacuum device, and let washing buffer go through column
?. [Sample Analyse]
Add 6 mL of washing buffer 2, turn on vacuum device, and let washing buffer go through column
?. [Sample Analyse]
Add 12 mL of washing buffer 1, turn on vacuum device, and let washing buffer go through column
?. [Sample Analyse]
Add 1 drop of developing solution
?. [Analysis of the Result]
Wait 5 min to visualize reaction blue color = positive, white color = negative
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Alarm substance, or Schrekcstoff, is an mixture of compounds produced by specialized club cells in the epidermis of Ostariophysan fish. When lesioned, these cells release the substance, which acts as an aversive olfactory stimulus capable of evoking intense behavioral responses (alarm reaction) in other fish in the shoal (Maximino et al. 2018). Alarm substance is used as an aversive stimulus to study fear-like behavior in Ostariophysan - especially in zebrafish. The present protocol describes a technique to easily extract alarm substance for acute exposure.

[GUIDELINES]
Guidelines for humane euthanasia must be followed, and animals should NEVER be euthanized without deep anesthesia. See Matthews & Varga (doi: 10.1093/ilar.53.2.192) for a review.

[STEPS]
SECTION: Preparation
?. 
SECTION: Anesthesia
?. 
SECTION: Anesthesia
?. 
SECTION: Anesthesia
?. 
SECTION: Anesthesia
?. 
SECTION: Anesthesia
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?. 
SECTION: Alarm substance preparation
?.
TITLE: XRF Soil Sample Preparation
AUTHORS: rund.tawfiq

[DESCRIPTION]
This protocol outlines a procedure for the preparation of both wet and dry soil samples for X-ray Fluorescence (XRF) inorganic chemical analysis. The accurate analysis of soil samples is essential for understanding elemental composition and its implications for various environmental studies. Proper preparation ensures reliable and reproducible results.

For wet soil samples, the protocol encompasses drying to remove excess moisture, preventing potential interference with subsequent XRF analysis.

For wet and dry soil samples, the protocol outlines grinding to ensure homogeneity and fine particle size. Proper grinding enhances the accuracy of XRF analysis by promoting even distribution and facilitating the release of elemental components.

[STEPS]
1. Weigh ~500 mg of soil

2. Place soil in a sterile, heat-resistant vessel. Place the vessel on a hotplate at 50oC until soil is visibly dry (30 min - 1 hr).
3. Grind with a mortar and pestle until the soil turns to powder (1-2 mins)
2. Grind with a mortar and pestle until the soil turns to powder (1-2 mins)
TITLE: Bacterial Plating Quantification
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to serially dilute and plate cultured bacteria in suspension for quantification.</div></div>

[STEPS]
?. [Prepare Serial Dilutions]
Transfer of stock solution to 2 mL centrifuge tube.
100 µl
?. [Prepare Serial Dilutions]
Add DI water to the same centrifuge tube to make a dilution that is one-tenth the concentration of the stock solution.
900 µl
?. [Prepare Serial Dilutions]
Using of the dilution you just made, make another dilution that is one-tenth the concentration of the second solution.
100 µl
?. [Prepare Serial Dilutions]
Repeat 6 times until you have a solution that is diluted by 106.
?. [Plate]
Choose a dilution that you feel is appropriate and dispense of that dilution on agar plate.
100 µl
?. [Plate]
Evenly spread the cell suspension over the agarose gel with inoculation loop.
This may be different from how you have streaked before since this is for quantification, not identification. We are wanting the solution evenly spread so that we can count how many colonies are formed after incubation.
?. [Plate]
Wrap the agar plate's edge in parafilm.
If you have not worked with Parafilm, ask a TA for help in wrapping your plate.
?. [Plate]
Wait 2 minutes, then flip the plate upside-down for incubation.
?. [Plate]
Label the plate with:Bacterial strainDateDilution used (10^x)Initials
?. [Incubate]
Incubate at over night.
?. [Incubate]
Take image of incubated plate.
?. [Quantify Concentration]
Count the number of colonies formed. You can use the image of the plate. This can be done by hand or using an image processing software such as ImageJ.
?. [Quantify Concentration]
Calculate the original E. Coli concentration using the following equation:
TITLE: UC Davis - Triglyceride Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H2O2). In a Trinder5 type color reaction catalyzed by peroxidase, the H2O2 reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample.</div></div>

[STEPS]
?. Reconstitute powdered reagent with only 25 ml of distilled water to make a 2X solution.
?. Add 3 μl of calibrator and sample to each well.
?. Add 150 μl of PBS to each well. Read at 540 nm.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 150 μl of 2X reagent to each well. Incubate at 37°C for 5 minutes. Read at 540 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be
calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: 02 Enzyme Digestion
AUTHORS: TJUSLS China

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Mix together: 3μl  Restriction endonuclease 13μl  Restriction endonuclease 26μl  10xgreen buffer3000ng DNAAdd to 60μl dd H2OIf it is double enzyme digestion verification before sequencing:1μl  Restriction endonuclease 11μl  Restriction endonuclease 21μl  10xgreen buffer7μl  DNA
?. Heat in water bath or heat block at 37℃ for 30min-1h.
37 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The aim of this protocol is to describe how to do a very reproducible upshift of a nitrogen-limited yeast culture to nitrogen non-limited conditions. In order to do this with enough cells to make inefficient analysis (ie RATEseq) feasible, we start in the chemostat for high-cell counts.</p>

[GUIDELINES]
<p>This protocol contemplates a 200ml culture as the starting condition. Scaling it up a bit is simply a matter of multiplying things.</p>

[STEPS]
?. 
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