eamag/protocols-with-steps huggingface dataset:
TITLE: Bogus Sample Prep Protocol
AUTHORS: Abby   Moore

[DESCRIPTION]
This is a bogus protocol for assessing protocol development.

Here, I'll use the citation component to refer to a publication that might influence protocol development.


 


[BEFORE_START]
This is what you should know before you start the protocol ....

[GUIDELINES]
This is who's responsible for the protocol ....

[STEPS]
SECTION: This is my first section
1. Chill   to °C .  

I used the reagent and temperature components in this step.


SECTION: This is my first section
2. Combine 1 µL   and 1 µL   . 

I used the amount and reagent components in this step.

SECTION: This is my first section
3. Centrifuge the  using the following parameters  27000 rcf, 2 min, -20 °C .

I used the reagent and centrifuge components in this step.

I also use the centrifugation step icon.

SECTION: This is my second section
4. Dry the   for 1440 min using 
 

I used the reagent, duration equipment components.

I also used the overnight step icon.

SECTION: This is my second section
4.1. This is an arbitrary sub-step that tells you to   .

I used the GoTo component in this sub-step.

SECTION: This is my second section
5. 
 


I used the note component and the critical step icon.


SECTION: This is my second section
6. This is a step with special safety concerns.
 

I used the safety warning component and the toxic step icon.
SECTION: This is my second section
7. Use 50 % volume   in   to do something.

I used the volume and reagent components in this step.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the blunting protocol for NEB PCR Cloning Kit (E1202)

[BEFORE_START]
Reaction volume may be scaled up or down as necessary.

[GUIDELINES]
Reaction volume may be scaled up or down as necessary.&nbsp;<br /><br />PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.&nbsp;<br /><br />Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in&nbsp;<a href='https://www.neb.com/products/e1201-quick-blunting-kit'>Blunting Buffer</a>, but is also active in NEBuffers 1.1, 2.1, 3.1, and CutSmart&trade; Buffer in addition to NEBuffers 1-4, BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 and 1.1 where the total yield is about 50% of optimum.

[STEPS]
?. 
?. 
?. 
?.
TITLE: FIXATIVE FOR MOSQUITO LARVAE
AUTHORS: Rochelly da Silva Mesquita, Wanderli Pedro Tadei

[STEPS]
?. Accurately weigh  of borax and dissolve it in   of distilled water. Then, add  of the formaldehyde solution and  of glycerol, mix  and bring to volume of   with distilled water.
5 g
100 ml
10 ml
2.5 ml
1000 ml
TITLE: Phenotypic analysis of circulating leukocytes
AUTHORS: John Davis Coakley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Antibody panels for flow cytometry were prepared with specific antibodies for identification of lymphocyte populations, and antibodies specific to IL-23 receptor andIL-12 receptorβeta-2 subunit.</div><div class = "text-block">Single stains were used for compensation controls and fluorescence minus one (FMO) controls were used to help set gates for flow cytometry.</div></div>

[STEPS]
?. Acquire blood sample from patient - 3mls EDTA
?. Transfer 100mcl whole blood to each FACS tube (7 tubes per patient)
?. Add Dead Cell Stain into each tube except tube 7 (unstained), vortex, and incubate for 15 minutes in the dark
?. Resuspend with appropriate antibody cocktail (50mcl per tube) into each tube except 7 (unstained), vortex, and incubate for 15 minutes
?. Add 1ml BD FACS lysing solution, vortex, and incubate for 15 minutes in the dark
?. Add 1ml PBA and spin down at 1500RPM for 7 minutes. DIscard supernatants
?. Resuspend in 2mls PBA, vortex, and spin and discard as per step 6
?. Resuspend cells in few drops of PBA, vortex, keep cool, and acquire by flow cytometer immediately
?. Wash with PBA, vortex, and spin down at 1500RPM for 7 minutes
TITLE: Transmission Electron Microscopy of Native Nanodiscs
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This is a protocol for conducting transmission electron microscopy of native nanodiscs to determine population size distribution and morphology.

[STEPS]
SECTION: Staining grids
1. Dilute nanodisc samples into single molecule range, typically around between 1:10 and 1:100 is the samples have been through size exclusion chromatography using dilution buffer (50 millimolar (mM) .

SECTION: Staining grids
2. Glow-discharge carbon-coated copper grids (200 mesh) for 30 s seconds.

SECTION: Staining grids
3. Apply 5 µL to the grid and after 1 min minute blot off with Whiteman ashless filter paper.

SECTION: Staining grids
4. Wash grid once with 5 µL  for 5 s seconds.

SECTION: Staining grids
5. Stain grid with 5 µL for 1 min minute and blot dry with Whiteman ashless filter paper.

SECTION: Staining grids
6. Allow to dry for 30 min minutes to  
SECTION: Image Acquisition
7. Take micrographs using a JEOL JEM 1400PLUS electron microscope at an operating voltage of 80 kV.
TITLE: Somatic Signs of Withdrawal
AUTHORS: Lauren Smith, Nathan Rizo, Lani Tieu, Olivier George

[STEPS]
?. [Procedure]
Place two observation cylinders on a surface
?. [Procedure]
Place one rat in each cylinder
?. [Procedure]
Set a timer to 30 minutes, at which point both observers will begin to watch the animals
?. [Procedure]
Behaviors to be tallied:JumpsTeeth ChatteringPtosisBlinksHead ShakesPaw TremorsAbdominal ContractionsGenital LicksYawns
?. [Procedure]
Behaviors to be scored (0-3):Pilo erectionDiarrhea
?. [Procedure]
One tally is one behavior isolated by another behaviorExample: the animal chatters teeth for a while, then licks genital, then chatters teeth again_ that is two tallies for teeth chattering and one for genital licking
?. [Procedure]
Pay attention to the animal during the full 30 minutes- do not distract each other
?. [Procedure]
Sum the scores for each animal to get the withdrawal score
TITLE: Habituation and dishabituation (olfaction test)
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Habituation and dishabituation (olfaction test) for mice

[STEPS]
1.   Individualize each mouse in a different cage with new bedding. 
2. Present one cotton swab to the animal for object habituation for 10 min.
3. Present a second swab impregnated with water for 3 min.
4. Measure the number of times the animal goes towards the second cotton swab and the time the animal spends sniffing it.
5. Present a third swab impregnated with lemon essence (Essenciales) for 3 min. 
6. Measure the number of times the animal goes towards the third cotton swab and the time the animal spends sniffing it.
7. Calculate the discrimination index (DI) according to the formula: (Time exploring lemon essence – Time exploring water) / (Time exploring lemon essence + Time exploring water).
TITLE: Immunofluorescent labeling of cultured cells 
AUTHORS: Maryana Nissan, Divya.D.A Raj

[DESCRIPTION]
This protocol describes the process of Paraformaldehyde fixation [PFA-fixation] and immunofluorescent-labeling of cultured cells. 

[STEPS]
1. Wash cells with 1X Phosphate-Buffered solution [1X PBS].
2. Fixate cells in 4% PFA for 15 minutes at room temperature [RT]. 
3. Wash cells with 1X PBS twice, for 5 minutes each. Here, cells can be stored in 1X PBS at 4C for up to a week.
4. Permeabilize cells with 1x PBS + 0.1% TriotonX-100 for 5 minutes at RT. 
5. Wash with 1X PBS twice, for 5 minutes each wash at RT. 
6. Block with 10% serum (from species of secondary) for 1 hour at RT. 
7. Drain and incubate with primary antibody diluted in 5% serum overnight at 4C. 
8. Wash in 1X PBS thrice, 5 minutes each wash. 
9. Detect primary antibody with Alexa Fluor secondary antibody diluted 1:400 in 5% serum at RT for 1 hour in the dark.
10. Wash in 1X PBS thrice, for 5 minutes each wash at RT.
11. Add mounting media with DAPI and apply the coverslip.
12. Store slides in the dark at 4C.
TITLE: Tranfection of SpCas9 containing plasmids to generate cell lines with Cas9
AUTHORS: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2, Julius Lukeš1, 2, 3

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We electropoarted Spcas9 containing plasmid (pVY015) with </span><span style = "font-style:italic;">Leishmania mexicana</span><span> UTR under hygromycin selection marker as described in Protocol 7.</span></div></div>

[STEPS]
?. We electropoarted Spcas9 containing plasmid (pVY015) with Leishmania mexicana UTR under hygromycinselection marker as described in Protocol 7.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The protocol describes the transformation of the choanoflagellate <em>Salpingoeca rosetta</em> using a Nucleofector 4d Device and SF kit from Lonza. Plasmids that use <em>S. rosetta</em> promoters and regulatory elements to drive the expression of luciferase and fluorescent protein reporters are being deposited at Addgene. </p>

[BEFORE_START]
<p> </p>
<p>Place the following items in the biosafety cabinet to make the procedure proceed rapidly.</p>
<ul>
<li>4 x 50 ml conical tubes</li>
<li>1.5 ml centrifuge tubes</li>
<li>Artificial Seawater (ASW) filtered through a 0.22 µm filter</li>
<li>500 ml bottle to collect waste</li>
<li>Sterile fine-tip transfer pipettes</li>
<li>Serological pipettes</li>
<li>Aliquot of thawed pretreatment buffer from Protocol E</li>
<li>Pipettes and tips for transferring volumes of 1-1000 µl</li>
<li>Fine-tips (gel-loading tips) for transferring 100-200 µl volumes </li>
<li>Multichannel pipette for transferring 100 µl volumes</li>
<li>Reservoir for multichannel pipette if doing multiple samples</li>
<li>96-well nucleofection plate (Lonza V4SC-2096)</li>
<li>Papain stock solution (Step 3)</li>
<li>12 well plate(s)  (Corning 353225) containing 1 ml of 1% ASWC (See Step 1 for guidelines on culture media)</li>
</ul>
<p>Before you start the procedure, place the following items in an ice bucket next to the biosafety cabinet</p>
<ul>
<li>Recovery buffer from Protocol F</li>
<li>BSA from Protocol D</li>
<li>Reporter DNA mixture (Step 2)</li>
<li>SF Buffer (Lonza V4SC-2096)</li>
</ul>

[GUIDELINES]
<ol>
<li>To maintain sterility, perform all steps in a biosafety cabinet unless otherwise noted.</li>
<li>
<p>For protocols on starting and maintaining choanoflagellate cultures and preparing media, see King N, Young SL, Abedin M, Carr M, and  Leadbeater BSC (2009) <em>Emerging Model Organisms, Vol. 1, CSHL Press</em> and Levin TC and King N (2013) <em>Current Biology</em> 23:2176-80. </p>
</li>
<li>
<p>For seawater recipe, see Cembella AD (1995) <em>Manual on Harmful Marine Microalgae</em>, Chapter Three: Culture Methods. UNESCO (http://unesdoc.unesco.org/images/0013/001317/131711e.pdf)</p>
</li>
</ol>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Quantitative reverse transcriptase-PCR analysis
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

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

[STEPS]
?. Total RNA was purified using RNeasy™ (Qiagen, Valencia, CA, USA).
?. For cDNA synthesis, 1 µg of total RNA was reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen).
?. Real-time PCR was performed using QuantiTect SYBR green PCR kit (Qiagen). PCR primers were purchased from Qiagen.
?. Amplification and detection were performed using Rotor-Gene Q real-time PCR cycler (Qiagen).The change in expression of each target mRNA was calculated relative to the level of 18S rRNA.
TITLE:  Use of haemocytometer to quantify concentration of cells&#39; suspensions
AUTHORS: abotero  

[DESCRIPTION]
This protocol presents the procedures to estimate the concentration of cells' suspensions using an hemocytometer

[GUIDELINES]
The lower limit for accurate counting of cells in a hemocytometer is usually considered to be 2.5 x 105/ml. 

For cell numbers greater than 2.5 x 106/ml, it is generally recommended that the sample be diluted.

[STEPS]
1. Wash the hemocytometer with a washer bottle and dry it using a paper towel
2. Cover the hemocytometer with the coverslip and place it on a flat surface 
3. Vortex briefly the cell suspension whose concentration will be calculated
4. Using a micropipette collect 10-100 µl of the previously agitated suspension
5. Place the micropipette tip on the hemocytometer edge near the coverslip
6. Empty the spore suspension slowly into the chamber of the hemocytometer
7. Place the hemocytometer in the microscope's mechanical stage and secure it using the stage clip
8. Turn on the microscope and focus on the hemocytometer
9. Look out for the first square to be counted
10. Count the number of cells on each square
 

11. Count all the squares following a zig-zag 
 

12. Write down the number of cells on each square
 

 

13. Estimation of the concentration
 

13.1. If cells were counted on squares of size 1

Concentration (cells/ml)= Number of counted cells*10,000/Number of squares counted

 

13.2. If cells were counted on squares of size 2

Concentration (cells/ml)= Number of counted cells*160,000/Number of squares counted
 

13.3. If cells were counted on squares of size 3

Concentration (cells/ml)= Number of counted cells*250,000/Number of squares counted
TITLE: Osmolality-controlled fixation and simple preparation of human red blood cells for scanning electron microscopy
AUTHORS: Heather N Colvin, Glen Marrs, Regina Joice Cordy

[DESCRIPTION]
Scanning electron microscopy (SEM) provides a way to visualize red blood cell (RBC) morphology. Previous methods for human RBC fixation can induce osmotic-related changes to healthy RBCs, which can interfere with interpretation of biological morphological changes. In addition, traditional methods for fixation and dehydration of RBCs and associated SEM preparations involve multiple chemicals and time-intensive steps. Here, we provide a simplified protocol for human RBC fixation with careful control of osmolality. This protocol omits the use of sodium cacodylate, osmium tetroxide, and ethanol gradient dehydration steps, yet results in comparable outcomes.

[BEFORE_START]
In order to obtain the human blood needed for these studies, ethical clearance must first be obtained.

[STEPS]
2. Suspend human RBCs in osmolality-controlled buffer
 


    Add 25 µL of packed, washed RBCs to 500 µL osmolality-controlled buffer from Step #1

    Mix well with pipette, gently
6. Dilute fixed, washed RBCs in dH2O 
Mix RBC pellet well with pipette and add 5 µL of fixed RBC pellet to 995 µL dH2O to create at 0.5% hematocrit (hct) suspension of washed and glutaraldehyde-fixed RBCs in water
Mix well and add 100 µL0.5% hct glutaraldehyde-fixed RBC suspension to12mm round Poly-L-Lysine coated coverslide* on slide warmer
 

Dry slides on slide warmer for 60 °C30 min 



4. Fix RBCs with osmolality-controlled glutarldehyde

    Add 500 µL osmolality-controlled 2% glutaraldehyde solution from Step #3 slowly to well-mixed RBC suspension from Step #2.
    Invert gently in microcentrifuge tube
    Incubate at Room temperature in the dark for 30 min
 

5. Wash glutaraldehyde-fixed RBCs

Centrifuge fixed RBCs after completion of Step #4 at 900 x g, 5 min 
Remove supernatant without disturbing the RBC pellet
 

Add 1 mL dH2O and mix pellet well with pipette
Centrifuge cells again 900 x g, 5 min 
Remove supernatant without disturbing the RBC pellet




1. 
 


Make osmolality-controlled buffer 
Add 300 µL 1X PBS (pH 7.4) + 200 µL distilled water (dH2O) in microcentrifuge tube for 500uL total osmolality-controlled buffer solution

Mix well
 

3. Prepare osmolality-controlled 2% glutaraldehyde solution
 

Combine240 µL dH2O, 240 µL 1X PBS (pH 7.4), 20 µL 50% glutaraldehyde in conical tube and mix well by inverting
TITLE: LRRK2 expression and purification
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the expression of human LRRK2 in Expi293F cells and its in vitro purification.

[STEPS]
SECTION: LRRK2 expression and purification
1. Transfect the constructs encoding 3xFlag-LRRK2, 3xFlag-LRRK2(I2020T), 3xFlag-RCKW or 3xFlag-GFP-LRRK2 into Expi293F cells according to manufacturer instructions.
SECTION: LRRK2 expression and purification
2. Express the proteins for 4320 min following induction according to manufacturer instructions.

SECTION: LRRK2 expression and purification
3. Harvest the cells by centrifugation (400 x g, 4 min) and lyse by 3 freeze-thaw cycles in lysis buffer.
 

SECTION: LRRK2 expression and purification
4. Remove the cellular debris by centrifugation at 15000 x g for 60 min at 4 °C.
SECTION: LRRK2 expression and purification
5. Mix the clarified lysate with anti-FLAG M2 resin for 120 min while rotating at 4 °C.
 

SECTION: LRRK2 expression and purification
6. Wash the resin with 3x10 bed volumes of lysis buffer.
SECTION: LRRK2 expression and purification
7. Elute the proteins with lysis buffer supplemented with 0.2 mg/mL 3xFlag peptides.
 

SECTION: LRRK2 expression and purification
8. Remove the N-terminal 3xFlag tag by incubation with the GST tagged Prescission Protease (0.01 U/µL)   while rotating at 4 °C.

SECTION: LRRK2 expression and purification
9. Remove the GST tagged Prescission Protease subsequently by Glutathione Sepharose.
SECTION: LRRK2 expression and purification
10. Assess the purity of the proteins by SDS-PAGE and Western blotting.
SECTION: LRRK2 expression and purification
11. Dialyze the purified proteins   at 4 °C against the dialysis buffer.

SECTION: LRRK2 expression and purification
12. After dialysis, clarify the proteins by centrifugation at 17000 x g for 10 min at 4 °C.
SECTION: LRRK2 expression and purification
13. Determine the protein concentration by SDS-PAGE using Bovine Serum Albumin (BSA) as standard and used without freezing in liposome binding and tubulation experiments.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The protocol aims explicitly to amplify RRV viruses and not other viruses.</p>
<p>Alyssa Pyke designed the original assay which was published in 2011 (see below). Subsequently, Alyssa Pyke modified the reverse primer, and it is this most recent, revised version, described here.</p>
<p>The assay targets the E2 gene region and is designed as a qualitative test for investigating RRV infection of humans and arthropods.</p>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Fresh 4% Paraformaldehyde in PBS
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
Freshly prepared 4% Paraformaldehyde in PBS is used to fix adult and developing mouse brains during the transcardial perfusion process, as well as for immersion fixation.  This solution can be used for no more than one week post-preparation date.

Note: 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.

[STEPS]
TITLE: Gavage - food supplementation for stalled weight gain in mice
AUTHORS: taylor.panczyk

[DESCRIPTION]
In mice, specifically in Ndufs2fl/fl mice, weight gain usually stops above 70 days post injecton with the TH-Cre AAV into SNc. From that point, food supplements should be provided once daily via a gavage of 0.4-0.5 mL Ensure (Ensure original; 9gr protein,220 calories and we use Plastic feeding Tubes 22gax25mm FTP-22-25n from INSTECH mfg 2021-08-17; www.instechlabs.com) as long as mice keep gaining weight. When the second stop of weight gain happens, another daily gavage and 1 mL of subcutaneous saline should be added as necessary care.

[STEPS]
SECTION: Gavage
1. Provide light anesthesia with isoflurane.

  

SECTION: Gavage
2.  Once the animals are ready we look to put the feeding tube behind its tongue.

  

SECTION: Gavage
2.1. Once there, we place the feeding tube parallel to the animal with a light hyperextension, in this manner the feeding tube will get into the stomach with no effort**

*With this detail we will be avoiding all complicaBon for gavage that can happen acute or
chronic.

 
SECTION: Gavage
3. Slide the feeding tube in to the stomach and administer solution.
TITLE: Yale - Beta hydroxybutyrate (Cobas)
AUTHORS: Gary Cline, John Stack

[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 β-Hydroxybutyrate in blood, serum, and plasma. β-Hydroxybutyrate is measured by the oxidation to acetoacetate, followed by reduction of an indicator dye (monitored at 505 nm) by NADH.</div></div>

[STEPS]
?. Calibrate Cobas for β-Hydroxybutyrate by running a β-Hydroxybutyrate standard and three ß-Hydroxybutyrate controls.
?. Sample handling as performed by the Cobas Mira Plus.    a) Pipette 3 µL of sample into cuvette.    b) Add 105µL of ß-Hydroxybutyrate Reagent.    c) Mixture is incubated at 37˚C for 10 minutes.    d) Absorbance is measured at 505 nm.