eamag/protocols-with-steps huggingface dataset:
TITLE: PBMC Stimulation with Peptide Pools and Fluorospot Assay
AUTHORS: cecilia , Alessandro Sette

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to stimulate PBMCs with peptide pools for 14 days followed by Fluorospot assay.</div></div>

[STEPS]
?. [Thawing of Cryopreserved PBMC]
Add  (complete RPMI with 5% human serum) and  per vial of cells to a 50 ml tube.
[media]
[Benzonase]
?. [Thawing of Cryopreserved PBMC]
Place PBMC vials in  water bath for approximately 60-90s.
37 °C
?. [Thawing of Cryopreserved PBMC]
Before cells are completely thawed, add them to the prepared 50 ml tube.
?. [Thawing of Cryopreserved PBMC]
Centrifuge at .
Centrifuge: 1200 33, 7 min
?. [Thawing of Cryopreserved PBMC]
Pour off media and resuspend in media.
?. [Thawing of Cryopreserved PBMC]
Count cells and record viability (trypan blue).
?. [Thawing of Cryopreserved PBMC]
Centrifuge at .
Centrifuge: 1200 33, 7 min
?. [Thawing of Cryopreserved PBMC]
Pour off media and resuspend according to cell count to a final concentration of 4x106 cells/ml.
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
Resuspend thawed PBMC at 4x106/ml.
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
Add  per well and .
[cells]
[stimulus (5µg/ml final concentration)]
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
Add media supplemented with 10 U/mL (final concentration) of rIL-2 every 3-4 days.
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
Day 4 – add  supplemented with IL-2.
[media]
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
Day 7 and 11 – replace  supplemented with IL-2 without disturbing cells.
[media]
?. [Thawing of Cryopreserved PBMC]
Record peptide pool information:
?. [In vitro Restimulation for 14 days (Plate cells (2x106 per well) in 24-well plate)]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
We use this protocol to test if flies have preferance for darkness or light (phototactic or non-phototactic response).

[GUIDELINES]
We are using 3 day-old-flies (half of the experimental flies have their wings clipped, and half don&rsquo;t).<br /><br />

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Method to chemically bleach Aiptasia<em> </em>in small volume dishes.</p>
<p> </p>

[GUIDELINES]
<p>You should see a response by the 2nd day at the latest, with noticably whiter anemones and visible pellets of expelled algae in the water.  If not, sometimes the stock menthol solution loses effectiveness over time, so try increasing the dosage or making a new stock. Assess and repeat if needed.</p>
<p> </p>
<p>If algae are not being expelled fast enough after three days, you can try another menthol treatment or cold shock (though these may produce different bleaching effects). Cold shock consists of subjecting the animals to 4C for 4 hours per day for 1 week with abrupt water changes (rather than gradual cooling). This seems much more stressful to the animals, though they usually survive. </p>

[STEPS]
?. 
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?. 
?.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>per L:<br /></strong><br />20 g Sea Salts*<br />250 ml Basal Media <br />50 ml FeEDTA Stock <br />700 ml DIH<sub>2</sub>0 <br />0.1% vitamin supplement (optional)*<br /><br />*FeEDTA, carbon substrate and vitamins are added post-autoclaving once media has cooled to ~50&deg;C.<br /><br /><strong>Basal Media</strong> <br /><br />150 ml 1M Tris HCl pH 7.5 <br />87 mg K<sub>2</sub>HPO<sub>4</sub> <br />1.5 g NH<sub>4</sub>Cl <br />375 ml DIH<sub>2</sub>0<br /><br />(It's not a typo the total volume is 525 ml.) Autoclave and store at room temp.<br /><br /><strong>FeEDTA Stock Soln</strong> <br /><br />50 mg FeEDTA (Ethylenediamine tetraacetic acid; ferric-sodium salt)*<br />100 ml DIH<sub>2</sub>0<br /><br />Autoclave and store at room temp.<br /><br /><strong>Vitamin Supplement for Minimal Media<br /></strong><br />per 100ml:<br /><br />2 mg biotin <br />2 mg folic acid <br />10 mg pyridoxine-HCl <br />5 mg riboflavin <br />5 mg thiamine <br />5 mg nicotinc acid<br /> 5 mg pantothenic acid <br />0.1 mg cyanocobalamin <br />5 mg p -aminobenzoic acid<br /><br />Filter sterilize and store at 4&deg;C.<br /><br /><br /><br /><br />

[STEPS]
TITLE: Yale - Triglycerides
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of triglycerides in blood, serum, and plasma. Triglycerides are determined by coupling lipase, glucokinase, glycerol phosphate oxidase, and peroxidase to form a quinonemine dye which is measured at 500 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Triglyceride analysis by running a multi-analyte calibrator and two control serum.
?. Sample handling as performed by Cobas Mira Plus.     a) Pipette 4 µL of sample into cuvette.    b) Add 275 µL of Triglyceride liquid reagent.    c) Incubate at 37ºC for 10 minutes.    d) Absorbance is measured at 500nm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Gene specific primers are retrieved from Primer Premier 6.0 and National Center for Biotechnology Information Software；Fluorescent dye are SYBR® Premix Ex Taq<sup>™ </sup>II (Tli RNaseH Plus)(TaKaRa,China).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Make potassium orthophosphate solution
AUTHORS: Gurdon Institute media kitchen

[STEPS]
?. 
?. ABCDEF1SolutionPotassium
  Orthophosphates  2Strength0.17M &amp; 0.72pH: Batch size1.2L3Date
  of preparation    45IngredientsQuantityAdded (tick)Manufacturer/Batch No.6KH2PO4&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;( potassium
  dihydrogen orthophosphate)27.7g   7K2HPO4&nbsp;&nbsp;&nbsp; (dipotassium hydrogen orthophosphate)150.5g   8Double
  distilled H2Oup to 1.2L
ABCDEF1SolutionPotassium
  Orthophosphates  2Strength0.17M &amp; 0.72pH: Batch size1.2L3Date
  of preparation    45IngredientsQuantityAdded (tick)Manufacturer/Batch No.6KH2PO4&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;( potassium
  dihydrogen orthophosphate)27.7g   7K2HPO4&nbsp;&nbsp;&nbsp; (dipotassium hydrogen orthophosphate)150.5g   8Double
  distilled H2Oup to 1.2L
?. ABCDEF1SolutionPotassium
  Orthophosphates  2Strength0.17M &amp; 0.72pH: Batch size1.2L3      45Ingredients  Quantity  6KH2PO4&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;(potassium dihydrogen orthophosphate) 27.7g  7K2HPO4&nbsp;&nbsp;&nbsp; (dipotassium
  hydrogen orthophosphate)150.5g 8Double
  distilled H2Oup to 1.2L 9  10      1112Method     131Using a 2L measuring cylinder add 500ml of double distilled H2O.142Dissolve solutes and make up to final volume with double
  distilled H2O153Dispense into 12 x 100ml bottles.&nbsp;164Label, date and autoclave.
ABCDEF1SolutionPotassium
  Orthophosphates  2Strength0.17M &amp; 0.72pH: Batch size1.2L3      45Ingredients  Quantity  6KH2PO4&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;(potassium dihydrogen orthophosphate) 27.7g  7K2HPO4&nbsp;&nbsp;&nbsp; (dipotassium
  hydrogen orthophosphate)150.5g 8Double
  distilled H2Oup to 1.2L 9  10      1112Method     131Using a 2L measuring cylinder add 500ml of double distilled H2O.142Dissolve solutes and make up to final volume with double
  distilled H2O153Dispense into 12 x 100ml bottles.&nbsp;164Label, date and autoclave.
TITLE: Brain image simulation protocol
AUTHORS: Bin Fu

[DESCRIPTION]
This protocol details the method for simulating a brain image with small and large features

[STEPS]
1. 1. Prepare negative control images where no fluorescent puncta were labelled. These images are considered as background images.
2. 2. Select one or several cropped images containing large aggregates in the library and apply a sigmoid function to the selected cropped image where x in the sigmoid function is determined by the distance to the large aggregate in the cropped image. 
3. Simulate small puncta with 2D gaussian distribution on a blank image (same image size as the negative control image), the number of which should be determined from real images. The sigma of puncta should be determined from real images as well. 
4. Add cropped large aggregates onto the background images at a random location. Then add the simulated puncta onto the background image to form a simulated image with both small and large features. 
5. Record position, intensity and background information per small puncta expect for those overlapping with the large aggregates in the simulated image and save the results.
6. Repeat step2 to 5 again for other background images
TITLE: S-Complete Medium
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Large quantities of C. elegans can be grown in a liquid medium. Liquid cultures of C. elegans are usually grown on S Medium using concentrated E. coliOP50 as a food source. This is the S-Complete medium recipe.


[STEPS]
1. 977 mLof S-Basal medium (Link below)
 

2. 1 mL of 5 mg/ mL cholesterol (dissolved in Et-OH)

3. 10 mL of 1 Molarity (M) Potassium Citrate Buffer, pH 6.0


4. 10 mLof Trace metals solution (sterile)
 


5. 3 mLof 1 Molarity (M)CaCl2 (sterile)


6. 3 mLof 1 Molarity (M) MgSO4 (sterile)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Transfusion-transmitted malaria, Donor prevalence and health worker knowledge and practices. Protocols for preparation of blood smears (Thick and thin films), reporting of malaria parasites, blood grouping and design of questionnaires.</p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
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TITLE: Recombinase polymerase amplification assay for detection of Mycobacterium ulcerans DNA
AUTHORS: Michael Frimpong, Hubert Senanu Ahor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This document describes the standard operating procedure for the application of the real time Mu-RPA assay with Exo probe system. The assay detects </span><span style = "font-style:italic;">M. ulcerans</span><span> DNA (IS</span><span style = "font-style:italic;">2404</span><span>) from clinical sample or culture suspension. The assay consists of recombinase polymerase amplification TwistDx Exo kit procedure. The reaction mix must be prepared in an environment free of DNA amplicons. Personal protective clothing (i.e. lab coats, gloves) must be used throughout the process. </span></div></div>

[STEPS]
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
For each sample, prepare a mastermix as follows: 2.1 μl of Mu_RPA F1 (10μM),    2.1 μl of Mu_RPA R2 (10μM), 0.6 μl of Mu-RPA P (10μM), 29.5 μl of Rehydration Buffer and 8.2 μl of dH2O.
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
Vortex to mix and spin down.
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
Transfer  of mix  to each TwistAmp exo reaction pellet. Mix by pipetting up and down until the entire pellet has been resuspended.
42 µl
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
Add  of the DNA template to the mix.
5 µl
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
For each sample, add  280 mM magnesium acetate and mix well.
2 µl
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
Insert the tubes in Axxin T8 Isothermal device and run at the set program.
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
After 4 minutes, take the samples out and vortex to mix , spin down and return the samples to the T8 device.
?. [1.	Performing the amplification: Rehydration of reaction pellets and ‘magnesium start’]
Continue the incubation/detection for a total incubation time of 15 minutes.
TITLE: Hydra Medium 4.0
AUTHORS: Rob Steele

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the recipe for the medium that the Steele Lab currently uses for culturing all of its Hydra strains. A detailed description of the medium is in the following document: </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Fill Tank]
Fill Nalgene tank with 100 liters of high purity water (Nanopure, MilliQ, or equivalent). The volume markings on the side of the tank are accurate enough for determining how much water you have added.
?. [Add ingredients]
Add sodium bicarbonate Mix well with plastic paddle
4.2 g
?. [Add ingredients]
Add potassium chlorideMix well with plastic paddle
0.22 g
?. [Add ingredients]
Add anhydrous magnesium sulfateMix well with plastic paddle
3.97 g
?. [Add ingredients]
Add calcium chloride dihydrate Mix well with plastic paddle
14.7 g
TITLE: Fixing cell pellets
AUTHORS: Verity Goodwin, Emily Souster, Mathew Garnett, Fiona Behan, Charlotte Beaver, Rizwan Ansari, Adam Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to outline the process of fixing cell pellets in 1.5ml tubes. It has been developed within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute.</div><div class = "text-block"><span style = "font-weight:bold;">Process diagram:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/11f7e279594fdb8947a627a79a011af77912ade8923993cae1dcb9e618960e5f/ccmmbdtbp.png" /></div></div>

[STEPS]
?. [Fixing]
In a fume hood, prepare a solution of 3.7% formaldehyde in PBS
Chemical safety: Formaldehyde 37% and 3.7% must be prepared and used only in the chemical fume hood, using chemical resistant gloves. Waste must be kept in the fume hood and disposed of appropriately.
?. [Fixing]
In the fume hood, tap the tube to loosen the pellet and resuspend each pellet in   of 3.7% formaldehyde. Mix well by pipetting up and down, ideally using a Rainin 1ml electronic pipette to obtain a single cell suspension and incubate at room temperate to fix for 10-20 minutes.
500 µl
?. [Fixing]
Centrifuge cells at  using a minifuge inside the fume hood.
Centrifuge: 300 34, 3 min
?. [Fixing]
Carefully remove the supernatent (dispose of waste formaldehyde appropriately) and resuspend the cell pellet in   PBS and transfer to a 5ml BD Falcon Round Bottom Polystyrene Tube by pipetting through its filtered cap.
500 µl
At this stage, the fixed cell suspension can either be stored at   overnight or analysed using flow cytometry immediately.
TITLE: 10X TBS Buffer
AUTHORS: Bryon Drown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tris buffered saline (TBS) is a commonly used buffer with a wide range of applications in biochemistry. It is often more convenient to prepare a 10X solution so that additional components can be added when preparing the buffer on demand.</div></div>

[STEPS]
?. Weigh out Tris base
3.13 g
?. Weigh out Tris HCl
27.45 g
?. Weigh out sodium chloride
88 g
?. Dissolve in MilliQ water
900 ml
?. Measure and adjust to  with concentrated HCl and/or NaOH
?. Add MilliQ water to a final volume of 1 L
TITLE: Sensitivity test of toehold
AUTHORS: Hung Liang Pai, Cheng-Ruei Yang

[STEPS]
?. [Preparation]
Thaw the reagents including 1. DNase/ RNase free water (store in -20°C) 2. solution A (store in -80°C) 3. solution B (store in -80°C) 4. RNase inhibitor (store in -20°C) 5.toehold switches with invertase DNA (store in 4°C) 6.miRNA as the trigger (store in 4°C)
on ice
?. [Preparation]
Sterilize the bench, and put on a labmat
?. [Protocols]
Gently shake and spin down the tube before adding (especially solution A and DNA plasmid)
?. [Protocols]
Add below reagents in to PCR tubes in order:1. ddH2O(till totally )2. solution A3. solution B4. inhibitor5. toehold switches with invertase DNA6. miRNA or (depends on whether its control group or test group)
5 µl
2 µl
1.5 µl
0.2 µl
50 ng
50 ng
0 ng
?. [Protocols]
Centrifuge
Centrifuge: 4000 33
?. [Protocols]
Place the PCR tubes in the dry bath incubator
37 °C
?. [Protocols]
Put the PCR tubes   after 2 hours
on ice
?. [Protocols]
Put the PCR tubes in the dry bath incubator
55 °C
?. [Protocols]
Measure the glucose concentrations of each PCR tubes by glucometer
?. [Protocols]
Measure the glucose concentrations of each PCR tubes by glucometer again
TITLE: Collection and shipment of specimen for Visium Spatial Transcriptomics (vST/Visium ST)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment procedures for Visium Spatial Transcriptomics (vST/Visium ST).

[STEPS]
SECTION: Reagents and Materials:
1. 10% NBF fixative
Tweezers
Appropriate container for fixing
SECTION: Quality Key Points:
2. The tissue specimen should be always kept at 4 degrees Celsius and RNase-free. 
It is crucial to not store the tissue specimen at RT to avoid any cell death, and tissue and/or RNA degradation. 
SECTION: Procedure:
3. Collection/Harvest:
SECTION: Procedure:
3.1. Animal was euthanized via cervical dislocation. 

SECTION: Procedure:
3.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest. 
SECTION: Procedure:
3.3. Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
3.4. Heart, pancreas, and kidney was collected and the specimen was fixed in 10-20 X volume of 10% NBF fixative for 24-48 hours at room temperature.
SECTION: Procedure:
3.5. Samples were kept at 4 degrees Celsius until submission to histology for processing and embedding.
SECTION: Procedure:
4. Embedding:
SECTION: Procedure:
4.1. Samples to be embedded into FFPE blocks at Histology, Jax-BH.
SECTION: Procedure:
4.2. Samples can be stored with Histology at Jax-BH (Rama/Juliana will coordinate further procedures)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a collection of G-Biosciences protocols: SpinOUT™ for Desalting &amp; Buffer Exchange of Peptide &amp; Protein Samples.</p>
<p>(Cat. # 786-170 to 786-173, 786-703 to 786-708, 786-865 to 786-869)</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Heat dry block at 65o C<br /><br />Fly Griding Buffer (Adjust pH to 9.2)<br />
<table style="height: 161px;" width="535">
<tbody>
<tr>
<td> </td>
<td>For 100 mL</td>
</tr>
<tr>
<td>0.1M NaCl</td>
<td>584 mg</td>
</tr>
<tr>
<td>0.2M Sucrose</td>
<td>6.85g</td>
</tr>
<tr>
<td>
<p>0.1M Tris-HCl pH 9.1</p>
</td>
<td>1.21g</td>
</tr>
<tr>
<td>0.05M EDTA</td>
<td>1.861</td>
</tr>
<tr>
<td>0.5% SDS</td>
<td>5mL 10% SDS</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
?. 
?.
TITLE: Breast tumours dissociation 
AUTHORS: Samah El Ghamrasni

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol designed to dissociate fresh breast tissues (surgical specimens and biopsies) for single-cell RNAseq. 
</div><div class = "text-block">The protocol has been demonstrated to work successfully with fresh and cryopreserved tissues.</div></div>

[STEPS]
?. [Tissue Dissociation]
Transfer the tissue onto a 10 cm petri dish
?. [Tissue Dissociation]
Rinse 1x briefly with ice cold PBS and aspirate PBS off.
?. [Tissue Dissociation]
Use a blade to carefully cut the sample into small pieces, approximately 3-4 mm in diameter.
?. [Tissue Dissociation]
Transfer pieces into 50ml tube and Resuspend in 5ml of Base media + Liberase (200ug/ml)
?. [Tissue Dissociation]
Incubate 2 hours at 37C
?. [Tissue Dissociation]
Mix with a 5 ml serological pipet 5 times to break up the pieces.
?. [Tissue Dissociation]
Let the pellet settle at the bottom of the tube, and transfer supernatant to a new falcon tube (Filter supernatant using 40um mesh)
?. [Tissue Dissociation]
Resuspend remaining tissue in 2mL of TryplE, incubate for 10 min at 37C
?. [Tissue Dissociation]
Repeat Step 6 and 7
?. [Tissue Dissociation]
Spin cells down (300g for 10min at 4C) and Resuspend cells in resuspension media
?. [Tissue Dissociation]
Count cells
?. [Flow Sorting]
Stain cells with SytoxBlue (1ul/ml)
?. [Flow Sorting]
Incubate at room temperature for 15 min
?. [Flow Sorting]
Transfer to ice and proceed to flow sorting
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Purpose: This protocol describes the use of ImageJ to measure dimensions of viruses in TEM micrographs, but can be applied to the measurement of anything in any image. <br /><br />The ImageJ program can be downloaded free (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) and it&rsquo;s usage should be cited in publications using the reference listed on the website.

[STEPS]
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TITLE: Panda Express Keto: 100% Effective and Natural Ingredients!
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[DESCRIPTION]
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[STEPS]