eamag/protocols-with-steps huggingface dataset:
TITLE: Determination of NM Concentration
AUTHORS: Xiqun Chen

[DESCRIPTION]
This is the protocol for determining neuromelanin concentration and data.

[STEPS]
1. Place SNpc tissue in a plastic tube and carefully ground it. Weigh 10 mg of tissue for each sample, and place itin a 5 mL glass tube.
2. In each tube, add 1.5 mL of pH 7.4 phosphate buffer (50mM), shake, and centrifuge at 9000 x g for 30 mins. Discard the supernatant.
3. Wash with phosphate buffer and repeat once more.
4. Add 1.5ml of Tris buffer (50 mM, pH 7.4) solution, containing sodium dodecyl sulfate (5 mg/ml) and 0.2 mg/ml proteinase K to the pellet of each sample. Incubate the pellet by shaking in this solution for 2 hours at 37ºC.
5. Centrifuge the suspension of pigment at 9000 x g for 30 minutes.
6. Wash the pellet with 1.5 ml of NaCl solution (9 mg/ml) and 1.5 ml of water. Centrifuge at 9000 x g for 30 minutes.
7. Dissolve the NM residue in 1 ml of 1M NaOH at 80ºC for 1 hour.
8. Centrifuge this solution and transfer the supernatant into a quartz cuvette, measure the absorbance at 350 nm.
9. To run calibration curves dissolve known amounts of NM (ranging from 1 – 30 µg) in 1 ml of 1 M NaOH at 80ºC for 1 hour.
10. NM value was the average from 2-3 replicates. The final values of NM concentrations are expressed as µg/mg dry tissue.
TITLE: Cell culture 
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains the cell culture and characterization of lines ND1014, N1, and ND27760 from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [Cell Culture]
Obtain human iPSC lines from Coriell Cell Repositories or through reprogramming patient cells.
?. [Cell Culture]
Characterize pluripotency of iPSCs by immunochemistry for pluripotent cell markers (NANOG, OCT4, TRA‐1‐60, and SSEA‐3) and through embryoid body formation assay.
?. [Cell Culture]
Maintain iPSCs as feeder‐free cultures in mTESR1 medium in 5% CO2/95% air condition at  and passage using ReLeSR.
37 °C
?. [Cell Culture]
Perform karyotype analysis of G‐banded metaphase chromosomes to confirm the chromosomal integrity of iPSCs.
?. [Cell Culture]
Maintain iPSCs as feeder‐free cultures in mTESR1 medium in 5% CO2/95% air condition at  and passage using ReLeSR.
37 °C
?. [Cell Culture]
Characterize pluripotency of iPSCs by immunochemistry for pluripotent cell markers (NANOG, OCT4, TRA‐1‐60, and SSEA‐3) and through embryoid body formation assay.
?. [Cell Culture]
Perform karyotype analysis of G‐banded metaphase chromosomes to confirm the chromosomal integrity of iPSCs.
TITLE: BrainSaw 50 mM pH 7.4 PB slicing buffer
AUTHORS: Rob Campbell

[DESCRIPTION]
This protocol makes 2L of pH 7.4 phosphate buffer. This solution is used for serial section 2-photon imaging. 

[STEPS]
1. Fill 2L flask with MQ water to the  2L notch. Place on magnetic stirrer and set to 1000 RPM. 
2. Weigh 3.1 g of   and pour into the flask. 

3. Weigh out 20.8 g of   but do not pour into flask until existing solute is dissolved.   

4. Wait until    has dissolved. Skipping this step will result in a small quantity of fine precipitate being formed. 

5. Pour   weighed out above into flask. Screw on lid and shake once to ensure no solute is stuck around the rim of the flask. Return to stirring plate at 1000 RPM. 
6. Use PB once all solute is dissolved. Do not refrigerate: cold PB will form bubbles as it warms and this interferes with imaging.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>There has been an increased use of medical <em>Cannabis</em> in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers.  We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids.  Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.</p>

[STEPS]
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TITLE: Trypan blue and Turk solution
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Emanuela Rasini, Massimiliano Legnaro, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Trypan Blue test</span></div><div class = "text-block">Trypan is test used as a stain to selectively colour dead cells. Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells appear as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a dye exclusion method.Trypan is test used as a stain to selectively colour dead cells. Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells appear as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a dye exclusion method.</div><div class = "text-block"><span style = "font-weight:bold;">Türk's solution</span><span style = "font-weight:bold;"></span></div><div class = "text-block">Türk's solution is a hematological stain (crystal violet or aqueous methylene blue) that destroys the membrane of red blood cells (RBCs), white blood cells (WBCs) and platelets and platelets within a blood sample, and stains the nuclei of the white blood cells, making them easier to see and count. Turk's solution is a gold standard for counting leukocytes in a defined volume of blood.</div></div>

[STEPS]
TITLE: GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
AUTHORS: Tina.Pfefer, Julie Haendiges, Maria  Balkey, Ruth Timme

[DESCRIPTION]
Here we have created a collection of all the protocols used for WGS using the MiSeq, in order, from sample extraction to NCBI submission. 

This collection has three sections:

Section 1: WGS Wet lab workflow for Illumina MiSeq
Section 2: Dry lab workflow for sequence QC and NCBI submission - Direct Submission
Section 3: Dry lab workflow for sequence QC and NCBI submission - PulseNet labs


Associated protocols:

Querying the NCBI database for GenomeTrakr data (protocols.io)
NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)


[STEPS]
SECTION: Wet lab
1. WGS Wet lab workflow for Illumina MiSeq
SECTION: Wet lab
2. DNA Extraction
 

SECTION: Wet lab
3. DNA Quantification
 

SECTION: Wet lab
4. Library Preparation


 

SECTION: Wet lab
5. Sequencing
 

SECTION: Dry lab - Direct Submission
6. Dry lab workflow for sequence QC and NCBI submission - Direct Submission: 


The following protocols are also included in a Springer Methods book chapter collection: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback (protocols.io)

SECTION: Dry lab - Direct Submission
7. Check sequence quality:
 


SECTION: Dry lab - Direct Submission
8. Populate BioSample AND SRA metadata templates:


 



SECTION: Dry lab - Direct Submission
9. Submit sequence and metadata to NCBI:


 

SECTION: Dry lab - Direct Submission
10. Update, retract, or replace these records in NCBI databases, if necessary:

 

SECTION: Dry lab - PulseNet submission
11. Dry lab workflow for sequence QC and NCBI submission - PulseNet labs:
SECTION: Dry lab - PulseNet submission
12. Check sequence quality:

Follow the CDC's guidance for assessing QC in BioNumerics
SECTION: Dry lab - PulseNet submission
13. Populate NCBI template: 

***NOTE: For NCBI submission for fields not included in the BioNumerics Template, please remember to include the name of the laboratory sequencing the isolates and the surveillance effort name in the sequenced_by and project_name fields as described in Step 5 of this protocol.
 

SECTION: Dry lab - PulseNet submission
14. Submit sequence and metadata to NCBI:

Follow the CDC's guidance for NCBI submissions through BioNumerics.
SECTION: Dry lab - PulseNet submission
15. Update, retract, or replace these records in NCBI databases, if necessary:
TITLE: Hybridization of DNA oligos
AUTHORS: Laura Sánchez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to hibridate two complementary DNA chains.</div></div>

[STEPS]
?. [Annealing of oligonucleotides]
Add the required volume of H2O to the lyophilized oligonucleotides to obtain a concentration of 100 μM. Vortex both tubes for 30 s and incubate them at RT for 5 min to dissolve them.
?. [Annealing of oligonucleotides]
Prepare the annealing mix by adding into a PCR tube:>  45.5 μl of H2O>  2.5 μl of 1.0 M NaCl>  1 μl of oligo 5' (100 μM) >  1 μl of the oligo 3' (100 μM)
?. [Annealing of oligonucleotides]
Place the PCR tube with the mix in a thermocycler with the following annealing programme: -  5 min at 95°C-  1 min at 95°C -  ramp down 1°C per cycle for 72 cycles -  end by keeping the temperature at 10°C
?. [Annealing of oligonucleotides]
Take 10 μl of the annealed oligonucleotides and dilute it with 90 μl of H2O to obtain a 0.2 μM concentration.
?. [Annealing of oligonucleotides]
The annealed oligonucleotides stocks can be stored at −20°C for future use.
TITLE: Primary astrocyte culture
AUTHORS: Xiqun Chen, Qing Ye

[DESCRIPTION]
Primary astrocytes were obtained from C57BL/6 mice embryonic day 17. The dissected cortical tissue was digested, triturated, and centrifuged. The cell pellet was resuspended in high-glucose DMEM/F12 supplemented with 10% FBS. Cells were seeded in poly-L-ornithine-coated Petri plates. Nonadherent cells were removed after 5-7 days. 

[STEPS]
1. For primary astrocyte culture - Use C57BL/6J mice at embryonic day 17 
2. Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex. 
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
3. Collect the cortices in PBS  in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) on ice 
4. Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 15 min Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times 
5. Centrifuge the dissociated cortices (1500 rpm, 5 min) and resuspend the pellet in 10ml   medium supplemented with 10%  
7. Count the cells and plate them in a density of 50,000 cells/cm2 into PLL coated T-75 flask.
8. After 3 h, change the culture medium once and check the growth and survival of cells under the microscope.
9. Put the culture flask in a 37 °C-cell incubator for 1440 min

10. Change the media after 1440 min and then every 4320 min. A confluent layer of the astrocytes grows at the bottom in 5-7 days followed by a top layer of microglia.

11. After 5-7 days, put the flask on the shaker for 2 h at 200 rpm at 37℃ (incubator). Collect and discard the supernatant (consisting of microglia and some oligodendrocytes).
12. Wash the adherent cells with PBS and add 0.25% trypsin at 37℃ to detach the cells. 
Immediately add 10 mL DMEM medium containing 10% FBS

13. Centrifuge the cell suspension at 1500 rpm min for 5 min 

Resuspend the cells in a T75 flask or 24 well plate with high glucose DMEM supplemented with 10% FBS.

14. Identification of astrocytes by immunofluorescence staining: Use GFAP antibody (astrocytic marker) to identify the population of astroctes   
15.  Transduction with BRAF (Optional) 
Astrocytes were transduced with BRAFV600E, or BRAFWT, or vector lentivirus plus 8 μg/ml polybrene for 24 h.
16. After transduction, the cells were cultured for 120 h in high-glucose DMEM/F12 (400ul/well) with or without FBS, and the medium as well as the cells were collected for subsequent experiments.
6. Triturate the cell suspension 10 times with a 1ml pipette
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Modified from Bigelow: NCMAM Center. Components to prepare 1 L of f/2 vitamin solution.</p>

[BEFORE_START]
<p>Prepare primary stock solutions (in dH<sub>2</sub>O) of the following:</p>
<ul>
<li>biotin 0.1 g/L</li>
<li>cyanocobalamin: 1.0 g/L</li>
</ul>
<p>Filter sterilize through a 0.2 micron PES membrane and store frozen in 1 mL aliquots.</p>

[GUIDELINES]
<p>First, prepare primary stock solutions. To prepare final solution, begin with 950 mL of dH2O (autoclaved), dissolve the thiamine, add the amounts of the primary stocks indicated, and bring final volume to 1.0 liter with dH2O. Filter sterilize. Store in refrigerator or freezer.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Maddie Denney (mdenney5@vols.utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from Guan, Riezman, Wenk &amp; Riezman, 2010</p>
<p> </p>
<p>Please note that there are two versions of this protocol. Use the one that corresponds to your sample.</p>

[STEPS]
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TITLE: Approved Science Keto Reviews - Is It legitimate Or Fake?
AUTHORS: approvedsciencereviews  

[DESCRIPTION]
Approved Science Keto Reviews - Is It legitimate Or Fake?


[STEPS]
1. Approved Science Keto Reviews - Is It legitimate Or Fake?
Currently everyone loves junk food and some people are consuming lots of unhealthy food constantly. But this habit can take you towards rotundity and it's the problem which is being faced by millions in the USAalone.However, also we've an effective product for you, If you're looking for a natural result for dealing with fat. Approved Science Keto Capsules andDr. Keto Pills is the item in the request that's giving you a slim body with the help of the ketosis process. It's able of perfecting several body functions and you can get amazing health without doing any deep drill. Read the entire review for details. 
What's Approved Science Keto Capsules and Dr. Keto Capsules? 
Approved Science Keto Capsules and Dr. Keto Pills is a herbal mix for adding the speed of weight loss. You can end all your problems related to fat with the help of this product. It's able of promoting the ketosis system by reducing your appetite. It'll give you a better metabolic rate so that you can reuse food in a better way. Your bettered digestive system will help in reducing the poisons and dangerous waste in your body. It'll also help the storehouse of calories in your body for a long time. You can stop the product of fat cells in your body by consuming this item every day. 
Approved Science Keto Capsules and Dr Keto Pills is giving you high impunity and stamina to fight with several other conditions that are connected to rotundity. You'll be burning your body fat for producing energy and it's the reason for your high stamina. You can regulate your blood pressure and blood sugar situations with the help of this item. It'll minimize the threat of heart attack and you can save lots of plutocrat for yourself by perfecting your heart health. 
How does Approved Science Keto Capsules and Dr Keto Capsules work? 
This product starts working from the first day and it'll start by dwindling your appetite. Your stomach will feel full for a longer duration. Approved Science Keto Capsules and Dr Keto Pills is reducing the number of carbohydrates so that you can enter the state of ketosis easily. When your body isn't having enough carbohydrates, it starts using body fat for the product of energy. But your body is formerly in the habit of using carbohydrates as a source of energy. 

Official Website:- https://freaknewswire.com/index.php/2022/05/12/approved-science-keto-reviews-is-it-really-works/
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>MATERIALS<br /><br />Medium</strong>: NO LIF ES medium<br /><strong>Dish</strong>: BD Petri Dish.<br />Trypsin<br />4% PFA <br />PBS

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A description of how samples are collected and processed as part of MLML Smith Lab's weekly HAB monitoring efforts in Monterey, CA.</p>
<ul>
<li>Sampling Location: Monterey Commercial Wharf, Monterey, CA. 36° 36.3' N 121° 53.3' W</li>
<li>Oceanographic and meteorological observations for the time of sampling are referenced from the Monterey, CA NOAA station ID: 9413450</li>
</ul>

[STEPS]
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TITLE: Protocolo Tcg4
AUTHORS: dgalvana  

[DESCRIPTION]
Protocolo para realizar PCR de Tcg4

[STEPS]
SECTION: Protocolo de Tcg4
1. NOTA: Antes de comenzar es recomendable preparar la reacción sobre hielo. Todos los materiales deben estar estériles y limpios. Todo el procedimiento debe ser llevado a cabo dentro de la campana negra (previamente limpia con solución agua: cloro y esterilizada con UV durante 15 min)
SECTION: Protocolo de Tcg4
2. Prepare los tubos que habrá de utilizar para su PCR en una gradilla. NOTA: Preferentemente NO etiquete la tapa del tubo ya que se puede borrar, mejor etiquete en los costados
SECTION: Protocolo de Tcg4
3. Realice los cálculos de para preparar las reacciones necesarias (En este caso son 5 reacciones cada una con su control correspondiente, en total 10).
SECTION: Protocolo de Tcg4
4. Agregue cada componente de la tabla 1 en orden descendente en un tubo y marque como MasterMix (no agregar ADN ni primers). NOTA: coloque la polimersa al final. Saque del congeador la enzima y regresela inmediatamente después.

TABLA 1: Reactivos para PCR MasterMix

ReactivosUna reacción 10 reacciones Agua libre de nucleasas40.75 μL 407.5 μLBuffer 10x plus Mg5 μL50 μLdNTPs1 μL10 μLTaq polimerasa.25 μL2.5 μL
Se empelo el Kit PCR Reagent System

Primers2.5μL (1.25 μL de c/u)ADN molde.5 μL

SECTION: Protocolo de Tcg4
5. Coloque la cantidad correspondiente del MasterMixa cada uno de los tubos (47μL).
SECTION: Protocolo de Tcg4
6. Posteriormente coloque en cada tubo  los primers  y el ADN ha utilizar (volumen total de cada tubo 50 μL) NOTA: A los controles negativos no coloque  ADN, en su lugar agregue agua libre de nucleasas.
SECTION: Protocolo de Tcg4
7. Coloque los tubos en el termociclador ya programado con las condiciones de la PCR (ver tablas abajo).

Condiciones del Termociclador
 
PasosTemperaturaTiempoDesnaturalización inicial94 OC3minDesnaturalización94 OC45sHibridación53 OC30sExtensión72 OC1 minIncubación final72 OC10min 4 OCinfinito30 ciclos  


SECTION: Protocolo de Tcg4
8. Al terminar su PCR analice su producto en electroforesis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> </p>
<p> <strong> </strong></p>

[BEFORE_START]
<p><strong>Medium recipe:</strong></p>
<p>ATCC medium: 802 Sonneborn's Paramecium medium Solution 1 Rye grass Cerophyll:</p>
<p>Cerophyll*...................2.5 g</p>
<p>Distilled water..............1.0 L</p>
<p>Add cerophyll to distilled water and boil for 5 minutes.</p>
<p>Add 100 ml distilled water to compensate for evaporation.</p>
<p>Filter through Whatman #1 filter paper and add 0.5 g Na2HPO4.</p>
<p>Autoclave for 15 minutes at 121C.</p>
<p> </p>
<p><em>1. saltans</em> food (<em>K. pneumoniae</em>, or <em>E. coli</em>). I used only the <em>K. pneumoniae</em> so far</p>
<p> </p>
<p><strong>Agar Medium for <em>Klebsiella pneumoniae</em> ATCC-BAA-1705:</strong></p>
<p>Agar........................20.0 g</p>
<p> Yeast extract................4.0 g</p>
<p>Glucose......................0.16 g</p>
<p>Distilled water............800.0 ml</p>
<p>Dispense in 5 ml amounts. Autoclave for 25 minutes at 121C. Slant. Bacterium, grown on solution 2, is added to solution 1 (Just add very little, few colonies) and incubated at 30C for 24 hours prior to inoculation with Bodo saltans.</p>
<ul>
<li>Cerophyl powder that works best for the <em>saltans</em> is the powder from Pines.</li>
</ul>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>Culture Maintenance:</strong></p>
<ol>
<li>Prepare the bacterized <em>Bodo</em> <em>saltans </em>medium as described above. </li>
<li>Inoculate a T25 tissue culture flask (50ml) containing 20 to 25 ml of fresh medium with 1 to 2 ml from Bodo culture that is at or near peak density</li>
<li>Incubate horizontally at 18 to 22°C (room temperature can work fine) with cap screwed on not very tightly. 4.  Subculture every 7 to 10 days.</li>
<li>I usually subculture 3 to 4 flasks every week</li>
</ol>

[STEPS]
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TITLE: Labelled APIs
AUTHORS: BOC      Sciences

[DESCRIPTION]
Isotope-labeled API is a chemical reagent that contains one or several isotopic atoms in the molecule, and uses its traceability for analytical determination. The chemical properties of the reagents replaced by isotopes are usually unchanged except for isotopic effects. Therefore, isotope-labeled reagents can participate in similar chemical reactions and are widely used as tracers to study the laws of substance movement and change.

[STEPS]
TITLE: Cell Apoptosis Assay
AUTHORS: BOC       Sciences

[DESCRIPTION]
Understanding the mechanism of cell death and survival is the key content of toxicological analysis and drug development and application. Since the cell death pathway is complex and dynamic, multi-parameter analysis is very important for the accurate detection of this process.

BOC Sciences offers a wide range of apoptosis assays to measure multiple components on selectable testing platforms to accelerate your research.


[STEPS]
TITLE: U Cinn - Body Composition & Carcass Analysis
AUTHORS: Patrick Tso, Dana Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Total body composition in live, un-anaesthetized small animals and carcasses will reveal absolute amounts of body fat, lean tissue and body water via a quantitative magnetic resonance (QMR) instrument, EchoMRI, (Echo Medical Systems, LLC, Houston, TX). This instrument uses the differences in the nuclear magnetic resonance properties of hydrogen atoms in organic and non-organic environments to fractionate signals originating from fat, lean tissue and free water. </div></div>

[STEPS]
?. Insert the calibration tube into opening on right side of the EchoMRI-100 as far in as possible.
?. Select “Calibrate” at the bottom of the screen to calibrate the system.
?. After calibration has passed, weigh the animal and carefully place in the restrainer tube.
?. Insert the restrainer tube into the opening on the right side of the EchoMRI-100 and:   a. Select “New Experiment” at the bottom of the screen   b. Enter data for the Group,   c. Enter data for the Subject  d. Notes (Body weight should be included in the “Notes” field)
?. Select “Start Experiment” to start measuring the body composition. Each run will take approximately 1 minute.It is recommended that each animal is measured 2 or 3 times to determine the average of the repeated runs.
?. When the small box in the upper, left-hand corner reads “Experiment Complete,” remove the restrainer from the machine, and return the animal to its home cage.
?. Repeat steps 4-7 for all additional animals.