eamag/protocols-with-steps huggingface dataset:
TITLE: U Michigan - Hind Paw Withdrawal for Rodents
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol measures and quantifies the pain in rodent hind paw. These measurements can be used to evaluate the pathology present in the nervous system.</div></div>

[STEPS]
?. [Set up]
-  Must clean equipment with sporeklenz before entering the animal room
-  All restrainers need to be exclusively used in same room. If restrainer has been in another animal room, it CANNOT be used!
-  The True Tail Temp cannot be used for the paw test. It must be disabled. Press 6000E on keypad of CPU. It is on by default.
-  Test Head needs to be removed from Tail Flick machine and placed under heated glass.
-  Never remove cable from test head or back of tail flick machine when it is on. Failure to pay attention may damage electrical circuits in machine.
?. [Instrument Instructions:]
Keyboard commands ///There are 7 commands/// \\\number plus E shows the setting of the function\\\
1) #1 – Current Temperature (1E shows temp
2) #2–animalnumbere.g.2011Eforanimal11
3) #3 – Active Intensity (During Test) e.g. 3050E for 50% intensity
4) #4 – Idle Intensity (Temp during the ready state) e.g. 4050E for 50%
5) #5 – Cutoff timer (Harm prevention) e.g. 5025E for 25 seconds
6) #6 – Trigger Temperature (Pre-warming) ***disable***
7) #7 – Time of day (24 hr format{ 5 digit sequence}) e.g. 00100E for 1am8) #8 – Date (6 digits must be used) eg.020501 for Feb 5th, 2001
?. [Pre-Operating Instructions:]
- Mice need to be acclimated to the machine. This is accomplished with three 15 minute sessions per animal. The heat should be turned on medium (roughly equivalent to 32 Cº ̊) 10 minutes prior to when animals enter the box.
?. [Operating Insructions:]
-  For first animal type 6000E, 4001E, 3015E, 5015E, and set time and date
-  Place 1 mouse in each of the 6 cubes and let settle down for 15 minutes. Once calm, set the Test Head under the paw. Use idle intensity to focus the beam properly. Use as little idle intensity as possible during to avoid additional negative conditioning.
-  There are two ways to start test. The footswitch or the red button. Hit either one and the test will begin. When the mouse moves paw, press red button or footswitch to stop test.- When the test ends, there will be three numbers that flash   - P = Finish temp
  - B = Start temp
  - Blank = elapsed time
The time should be recorded only. Test is over
?. [Additional Notes:]
− Collecting the data can occur in two ways. After each test, the data can manually be collected or a printer can b set up to the machine and will print the data when there is one full page of data. Manual data collection is recommended at this point.
− Record 6 measurements. Alternate between left and right feet. Wait about 10 minutes between each test.
− Mice need to be totally calm. Data is not valid for excited mice.
−Settings to be used
   - Active Intensity is 25%
   - Idle Intensity is 1%
   - Cutoff time is 35 secondsIf the machine becomes confused, consult manual page 8, Troubleshooting.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A macrophage phagocytosis assay designed for use with recombinant, fluorescently labelled bacteria. This assay was optimised using recombinant GFP-labelled <em>E. coli</em> expressing eukaryotic-like proteins (ELPs) derived from sponge-symbionts.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Double Digestion of Insert DNA
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Double digest of insert DNA]
Combined enzyme volume should not exceed 1/10 of the total reaction volume.For double digestion with two FastDigest restriction enzymes, mix:  AB1ComponentAmount210x FastDigest Buffer2 µl3DNA400 ng4FastDigest Enzyme 11 µl5FastDigest Enzyme 21 µl6Sterile MilliQ WaterFill up with Sterile Milliq Water to 20 µl
AB1ComponentAmount210x FastDigest Buffer2 µl3DNA400 ng4FastDigest Enzyme 11 µl5FastDigest Enzyme 21 µl6Sterile MilliQ WaterFill up with Sterile Milliq Water to 20 µl
Total Volume can vary. Other components need to be adjusted.
?. [Double digest of insert DNA]
Mix gently and spin down
?. [Double digest of insert DNA]
Incubate at enzyme suitable conditions
Find out suitable conditions on website of particular science company
?. [Double digest of insert DNA]
Inactivate the enzyme at suitable conditions
Find out suitable conditions on website of particular science company
?. [Double digest of insert DNA]
Optional: Load on 1% agarose gel and run  (duration and voltage vary)
TITLE: Arabidopsis-Microbe interaction seed treatment
AUTHORS: Tao-Ho Chang

[DESCRIPTION]
The treated seeds can grow in medium or bulk soil to determine the impact of novel materials on plants. 

[BEFORE_START]
Seed priming is an important method that increases the health of the plant.

[GUIDELINES]
Seed treatment of arabidopsis seeds is a suitable, simple method for many plant-microbe interactions. 

[STEPS]
SECTION: Seeds sterilisation
1. The arabidopsis seeds are immersed with 50% bleach (2% Sodium hypochlorite) in 1.5 µL tubes.
SECTION: Seeds sterilisation
2. 
  


SECTION: Seeds sterilisation
3. 100 rpm, 10 s, 28 °C 

SECTION: Seeds sterilisation
4. Gently remove the supernatant and leave the seeds in the tube.
SECTION: Seeds sterilisation
5. The sterile seeds are immersed in distilled water.
SECTION: Seeds sterilisation
6.   
Repeat the process of step #3 to #5 for 10 times

SECTION: Seeds treatment
7. The seeds were immersed in the specific concentration of treatment
1. Bacteria treatment: OD value to 0.1 and diluted 10 times.
2. Fungus treatment: The final concentration of spore suspension is 100 spores mL-1.
SECTION: Seeds treatment
8.   

SECTION: Seeds treatment
9.   

SECTION: Wash seeds
10. The treated seeds are moved in a filter mesh (Favorgen) with a collection tube.
SECTION: Wash seeds
11. 100 rpm, 10 s, 28 °C 

SECTION: Wash seeds
12. Remove the filter through in collection tube
SECTION: Wash seeds
13. Add 500 µL of distilled water to the filter column and resuspend the seeds.
SECTION: Wash seeds
14.   
Repeat the process of step #11 to #13 for 10 times
TITLE: WATER PRODUCTION FOR AWARE (Metals)
AUTHORS: Celia Manaia

[DESCRIPTION]
The protocol summarises the procedures used for analytical control. The protocol describes the Standard Operating Procedure (SOP) for the optimization of advanced tertiary treatment of water, based on a comprehensive quality and risk assessment.

[GUIDELINES]
Directive 2008/105/EC of the European Parliament and of the Council of 16 December 2008 on environmental quality standards in the field of water policy.

[STEPS]
SECTION: Metals
1. The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage for720 min-1440 min(according to water load and season) and ozonation. The water quality is monitored at these three stages, for the parameters indicated in Figure 1 below.

 

SECTION: Metals
1.1. Sampling, Processing, and Analyses 

Water samples are collected (see Figure 2) and processed within a360 mininterval, before being shipped for the partner responsible for the analyses (Table 1). In case no processing is needed, samples are frozen and stored at-80 °Cwithin180 min.

For each sampling event, the date, day of the week and hour; the temperature and rain. Sampling points, indicated in Figure 2 were designated from A to I:
- Influent of primary treatment (A) 
- Influent of biological treatment (activated sludge) (B) 
- Treated secondary effluent (C) 
- Sand filter effluent (D) 
- UVC effluent (E) 
- Storage for reuse tank effluent (F) 
- Ozonation effluent (1 dose, e.g.,5 mgO3) - MITO3X technology - (G) 
- Effluent of the vacuum UV oxidation (VUV) (H) 
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I) 

 

SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2. Metals: 

Analysis: Detection and quantification of metals 

Method: Acidified water samples were analyzed for several metals (e.g. Al, Cu, Ni, Co, Pb, Zn, Cd, Pt, Sb, Sn, U, As, Ag, Hg, REEs) by means of ICP-MS (7900 Agilent) using matrix-matched external calibration. 

Observations: Samples were filtered/centrifuged within720 minafter collection, and then acidified to 0.15M HNO3.

SECTION: Parameters framed by Legal and Regulatory Requirements:
3. Using the EU Drinking Water Directive:  

Metals - DIRECTIVE 2008/105/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008 on environmental quality standards in the field of water policy
TITLE: High Molecular Weight (HMW) DNA Extraction Protocol for Tissue Sample
AUTHORS: Aswini Leela Loganathan

[DESCRIPTION]
This is an organic extraction protocol used for high molecular DNA extraction for tissue samples. This protocol is suitable for obtaining gDNA for PacBio Sequel IIe library preparation and sequencing.

[STEPS]
SECTION: HMW DNA Extraction Method
1. Lysis the tissue sample.
Add 500 µL of lysis buffer to 50 - 150 mg of Sample of sample. Make sure the tissue sample has been finely cut.

SECTION: HMW DNA Extraction Method
2. Denatures and digest proteins that are subsequently hydrolyzed with Proteinase K. 
Add 10 µL of Proteinase K and vortex for 10 s.

SECTION: HMW DNA Extraction Method
3. Incubate on a shaking incubator at 55 °C for 120 min ( or till dissolved). 

SECTION: HMW DNA Extraction Method
4. Remove RNA with RNAse .
Add 5 µL of RNAse and vortex briefly.

SECTION: HMW DNA Extraction Method
5. Incubate in a shaking incubator for at 37 °C.

SECTION: HMW DNA Extraction Method
6. Partitioning of lipids and debris into an organic phase using P:C:IA.
Add 500 µL of P:C:IA (25:24:1). Vortex until an emulsion is formed. 

SECTION: HMW DNA Extraction Method
7. Centrifuge at Room temperature for 10 min at 10000 x g.

SECTION: HMW DNA Extraction Method
8. Pipette 200 µL of the aqueous layer into a new tube.

SECTION: HMW DNA Extraction Method
9. Neutralize the charges on the sugar-phosphate backbone of the DNA with Sodium Acetate.
Add 1/10th :  20 µL 3M Sodium Acetate. Vortex gently (avoid creating bubbles). 

SECTION: HMW DNA Extraction Method
10. Precipitation Step.
Add 2 Vol : 440 µL of ice-cold 100% Ethanol. Mix gently and slowly by inverting the tube. 

SECTION: HMW DNA Extraction Method
11. Incubate at -20 °C for 120 min .

SECTION: HMW DNA Extraction Method
12. Centrifuge at 10000 x g, 2 min.

SECTION: HMW DNA Extraction Method
13. Wash Step.
Wash with ice-cold 70% ethanol. 
SECTION: HMW DNA Extraction Method
14. Centrifuge for 2 min at 10000 x g.

SECTION: HMW DNA Extraction Method
15. Repeat Wash Step (Step 13 and 14) 2 more times. 
SECTION: HMW DNA Extraction Method
16. Air dry for at least 30 min.

SECTION: HMW DNA Extraction Method
17. Final elution.
Add 35 µL of elution buffer (EB) and mix gently using a wide bore tips or by tapping.

SECTION: HMW DNA Extraction Method
18. Storage.
Store the gDNA at 4 °C
TITLE: Determination of viable cells by XTT
AUTHORS: tzhang 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the test method for analysis of metabolic activity in various cell lines with colourimetric XTT analysis. It can be used to determine cytotoxicity of various drug compounds.</div><div class = "text-block">XTT salt (2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) is a compound that is metabiolized by the purple dye formazan, in living cells, to form an orange colour. The colour change will only occur in the presence of these reagents and in living cells, so the amount of colour change can be used to quanitfy the amount of viable cells. The Beer-Lambert Law states that absorbance of a given wavelength will be proportional to concentration, and therefore the concentration of colour that is absorbed will be proportional to the number of viable cells.</div></div>

[STEPS]
?. Adhere cells to wells1.1 Add a specified amount of cells from stock to each well of the micro-well plate. Each well must have a consistent cell density.1.2 Incubate this plate at 37°C and 5% CO2. Allow cells to adhere to the well plate for 24 hours.1.3 Always include a control of untreated cells and a blank well with culture media with no cells.
?. Prepare the PMS and XTT solution2.1 Prepare the MPS solution by dissolving 3 mg PMS (Phenazine Methosulfate)  in 1mL of 1X PBS (Phosphate Buffered Saline).2.2 Prepare the XTT solution by dissolving 4 mg of XTT in 4 mL of the cell culture media used.2.3 XTT/PMS solutions can both be stored at -20°C in the absence of light. The expiry date will be 9 months from the date of preparation. Discard if there is evidence of growth, colour change, or recyrstallization.
?. Cell labelling with XTT3.1 Add 10 µL of the PMS solution to the 4 mL XTT solution to create the detection reagent.3.2 Add 50 µL of the XTT/PMS solution to each of the wells.3.3 Incubate the plate for 2-5 hours at 37 °C.
?. Sample Analysis4.1 Place the reaction on a shaker for a short period to mix the dye and detection reagent.4.2 Analyze the sample with a plate reader at λ= 450nm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The analysis of gene expression data via RNA sequencing (RNA-Seq) has become a standard method for model and non-model organisms to identify genes of interest associated with a specific stressor of physiological state. More recently, mass spectrometry (MS)-based proteomics started to become a standard method to assay the phenotype of an organism via means of its expressed proteins. MS has also become the method of choice for the study of protein phosphorylation. Phosphoproteomics targets proteins that are subjected to phosphorylation via means of their serine, threonine, and tyrosine residues. Phosphorylation is a reversible modification that plays a key role in controlling the activity of proteins as well as their function and subcellular localization.
To bring MS-based phosphoproteomics to the emerging coral model system Aiptasia, we developed a protocol for phosphosphproteins extraction and generation of a phosphoproteome assay (spectral) library, a comprehensive non-redundant collection of Aiptasia phosphoproteins. Based on the assay library, we then performed accurate label-free quantification (by DIA/SWATH-MS) of the Aiptasia phosphoproteome. Our method reproducibly identifies and quantifies a broad fraction of the phosphorylated proteome of Aiptasia and is comparable to methods developed for other model systems
 
 

[STEPS]
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?. 
SECTION: Methods - Reference phosphoproteome assay library generation (NOTE: This step is not done for phosphoproteome quantification)
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?. 
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Methods - MS acquisition for reference phosphoproteome assay library generation
?. 
SECTION: Materials
?. 
SECTION: Materials
?. 
SECTION: Methods - MS acquisition for phosphoproteome quantification
?. 
SECTION: Methods - Data analysis
?. 
SECTION: Methods - Reference phosphoproteome assay library generation / Phosphoproteome quantification
?.
TITLE: Open Field
AUTHORS: daniel.dautan, Per Svenningsson

[DESCRIPTION]
Behavioral assay for general locomotion in mice.

[STEPS]
1. Habituate naive mice to the room for 60 min. 
2. Transfer each mouse to a custom 40 cm x 40 cm dark box, with a clear floor and a light suspended on the center of the apparatus.
4. After each test and between each group, clean apparatus thoroughly with 50% ethanol followed by water.
5. Analysis: 
Center of open field is defined by the space located at least 5 cm from any given wall. 
Time in the center, distance travelled, and average speed is obtained by the built-in functions of the tracking software Ethovision XT11.
3. Test duration was set at 20 min.
TITLE: 10x Protocols: Visium Fresh Frozen Tissue Optimization -- University of Minnesota TMCs (CG000238 Rev E)
AUTHORS: 10x Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression on Fresh Frozen / OCT samples. Completed without CytAssist component.

1. Version of protocol used: 
   10x protocol CG000238, Revision E -- Tissue optimization required prior to taking tissue through Visium

2. Additional protocols added for guidance:
    10x protocol for imaging guidelines (CG000241, Rev D)
    10x protocol for tissue preparation guidelines (CG000240, Rev D)

Please note that these protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. 

[STEPS]
1. 10x protocol CG000238, Revision E (Tissue Optimization):
  
    For adipose: Determined Tissue permeabilization time = 15 minutes 
2. Additional protocols/guidelines
  CG000241, Rev D
  CG000240, Rev D
https://www.10xgenomics.com/support/spatial-gene-expression-fresh-frozen
TITLE: XPRIZE SHINE - Paper-based SARS-CoV-2 Saliva Test
AUTHORS: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to perform a SHINE paper-based assay to detect SARS-CoV-2 RNA from a self-collected saliva sample. This protocol is intended for in-home use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, </span><span style = "font-style:italic;">et al. </span><span>Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. </span><span style = "font-style:italic;">bioRxiv</span><span> (2020). </span></div></div>

[STEPS]
?. [Sample Collection and Viral Lysis]
Expel approximately one drop of saliva into the sample collection tube and cap the tube.    Saliva collection tube contains necessary volume of FastAmp® Viral and Cell Solution.
?. [Sample Collection and Viral Lysis]
Mix saliva sample and FastAmp® Viral and Cell Solution by shaking the closed sample collection tube for
?. [Sample Collection and Viral Lysis]
Wait incubating sample at  before proceeding to Step 4.
0 Room temperature
?. [SARS-CoV-2 Detection]
Add the entire volume in the dropper bottle containing Reagent Mix A to the tube containing lyophilized Reagent Mix B. Cap the tube.
?. [SARS-CoV-2 Detection]
Mix Reagent Mix A and B by shaking for approximately .
?. [SARS-CoV-2 Detection]
Dip the inoculation loop into the sample collection tube (a small layer of liquid should be contained within the loop).
?. [SARS-CoV-2 Detection]
Transfer liquid in inoculation loop to the Reagent Mix A and B tube by dipping the loop into tube and stirring for . Remove and discard the inoculation loop and cap the tube.
?. [SARS-CoV-2 Detection]
Mix the sample combined with Reagent Mix A and B by shaking for approximately .
?. [SARS-CoV-2 Detection]
Wait , incubating sample at , before proceeding to Step 10.
0 Room temperature
?. [Paper-based Readout and Automated Analysis]
Open the Sample-Reagent Mix A and B tube and place the test strip into the liquid with the arrows on the test strip pointing upward and towards you.
?. [Paper-based Readout and Automated Analysis]
Wait  with sample at  for visible horizontal bands to appear on the test strip.
0 Room temperature
?. [Paper-based Readout and Automated Analysis]
With the user-provided smart device such as a smartphone, open the HandLens application and select paper-based as the test type.
?. [Paper-based Readout and Automated Analysis]
Take a photo of the test strip, and select upload. The result of the test will appear on the smart device screen.
TITLE: Free Floating DAB Staining
AUTHORS: Ashley Harms, Jhodi Webster

[DESCRIPTION]
This protocol describes the procedure for performing 3,3'-Diaminobenzidine (DAB) immunohistochemical staining on free-floating tissue sections. The method is optimized to preserve tissue integrity and enhance antibody penetration by processing sections without being mounted to a slide. It results in a stable, permanent chromogenic precipitate for the microscopic visualization and analysis of antigen distribution.

[STEPS]
SECTION: DAY 1
1. Wash sections 3x5min in TBS
SECTION: DAY 1
2. Quench sections with solution for 5min at RT
Quenching solution (10mL – 3% H2O2 in 1XTBS):
4.5mL TBS 
4.5mL MeOH 
1mL 30% H2O2
SECTION: DAY 1
3. Wash 2x5min TBS
SECTION: DAY 1
4. Put sections in antigen retrieval for 30mins at 37C with agitation 
Antigen retrieval in TBS:     
10mM sodium citrate      
0.05% Tween-20
SECTION: DAY 1
5. Wash 3x5min in TBS
SECTION: DAY 1
6. Block in 5% serum + TBST for at least 1hr @ room temp
SECTION: DAY 1
7. 24-well plate (500μL/well): dilute primary Ab in TBST + 1% serum (of secondary host animal).
SECTION: DAY 1
8. Incubate overnight at 4C
SECTION: Day 2
9. Wash sections 3x10min in TBST
SECTION: Day 2
10. Incubate sections with diluted biotinylated secondary Ab [(1:1000) in TBST + 1% serum] for 2hrs at RT in the dark
SECTION: Day 2
11. Wash sections 3x10min in TBS in the dark
SECTION: Day 2
12. Dilute ABC solution (1:5 in TBS) and incubate sections for 30min at RT (using 2mL/well)
SECTION: Day 2
13. Wash 3x10mins in TBS
SECTION: Day 2
14. Follow DAB kit instructions (work in the hood, set up a bucket with bleach and water)
SECTION: Day 2
15. Prepare substrate working solution. To every 5mL of diH20, add:
SECTION: Day 2
16. 2 drops of Buffer Stock Solution
SECTION: Day 2
17. 4 drops of DAB Stock Solution
SECTION: Day 2
18. 2 drops of Hydrogen Peroxide Solution
SECTION: Day 2
19. If gray-black reaction is desired, add 2 drops of Nickel Solution
SECTION: Day 2
20. Move samples to substrate solutions and develop until sufficient colour change is evident; around 3-6mins
SECTION: Day 2
21. Immediately wash 3x5min in TBS
SECTION: Day 2
22. Mount sections on slides and dry for approximately 30min to 1hr or just until sections are adhered to slide.
SECTION: Day 2
23. Dehydrate:
70% ethanol 2x3min
90% ethanol 2x3min
95% ethanol 2x3min
100% ethanol 2x3min
Citrisolve 2x3min
SECTION: Day 2
24. Using Permount, cover with coverslip and dry several hours up to 2 days @RT.