eamag/protocols-with-steps huggingface dataset:
TITLE: MycoFluor Mycoplasma Detection 
AUTHORS: Ali Albalakhi, Ning Xia

[DESCRIPTION]
MycoFluor™ Mycoplasma Detection produces a fast and simple fluorescence microscopic assay that identifies mycoplasma infection in cell cultures. In order to detect mycoplasma, the fluorescent MycoFluor™ reagent is added to the culture medium, with or without cells, and the sample becomes stained and examined under a fluorescence microscope.

[STEPS]
SECTION: Protocol Testing of Culture Media
1. Take about 4mL of cell medium directly form the culture dish in which the cells have been growing centrifuge the sample at 1300 x g for 10min to pellet any cells and debris
SECTION: Protocol Testing of Culture Media
2. Carefully transfer 1mL of supernatant into labeled microcentrifuge tubes.
SECTION: Protocol Testing of Culture Media
3. Centrifuge the microcentrifuge tubes at 12,500 x g for 15 minutes
SECTION: Protocol Testing of Culture Media
4. Carefully remove and discard 0.5mL of supernatant, leaving behind 0.5mL of medium in the tube. Resuspend any pellet that may have formed using this 0.5mL of medium.
SECTION: Protocol Testing of Culture Media
5. Add 26µL of 20X concentrated MycoFluor reagent to 0.5mL of the medium.
SECTION: Protocol Testing of Culture Media
6. Pipet 10µL of the stained medium onto a clean microscope slide and cover with a clean coverslip.
SECTION: Protocol Testing of Culture Media
7. Seal the slide using quick dry clear nail polish topcoat by covering all sides of the coverslip.
SECTION: Protocol Testing of Culture Media
8. Image the slide using fluorescent microscope.
SECTION: Control Slides with Mycoplasma MORFS
9. Generating positive controls for the testing of culture media
SECTION: Control Slides with Mycoplasma MORFS
9.1. a. Pipet 5µL of the mycoplasma MORFS stock suspension on a clean, labeled microscope slide
SECTION: Control Slides with Mycoplasma MORFS
9.2. b. Add 5µL of stained medium to the slide 
SECTION: Control Slides with Mycoplasma MORFS
9.3. c. Cover with clear coverslip and seal using quick dry topcoat nail polish. 
SECTION: Control Slides with Mycoplasma MORFS
10. Image the positive controls and compare to the samples that have not been spiked with MORFS 
SECTION: Microscopy
11. Prepare the microscope with a near ultraviolet fluorescence filter (Excitation at 365nm and either bandpass 450nm ± 30nm or longpass >400nm emission filter)
SECTION: Microscopy
12. For optimum result, a 100X oil immersion objective is suggested but a 60X oil immersion objective will also work
SECTION: Microscopy
13. First look at the control slide spiked with MORFS to get an idea what it should look like. Then examine the test slide for extranuclear blue fluorescence.
TITLE: An 8-week resistance training protocol is effective in adapting quadriceps but not patellar tendon shear modulus 
AUTHORS: Pietro Mannarino, Liliam Fernandes Oliveira, Thiago Torres da Matta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Habitual loading and resistance training (RT) can lead to changes in muscle and tendon morphology as well as in its mechanical properties which can be measured by Shear Wave Elastography (SWE) technique. The objective of this study was to analyze the Vastus Lateralis (VL) and patellar tendon (PT) mechanical properties adaptations to an 8-week RT protocol using SWE. We submitted 15 untrained health young men to an 8-week RT directed for knee extensor mechanism. VL and PT shear modulus (µ) were assessed pre and post intervention with SWE. PT thickness (PTT), VL muscle thickness (VL MT) and knee extension torque (KT) were also measure pre and post intervention to ensure the RT efficiency. Significant increases were observed in VL MT and KT (pre= 2.40 ±0.40 cm and post= 2.63 ±0.35 cm, p = 0.0111, and pre= 294.66 ±73.98 Nm and post= 338.93 ±76.39 Nm, p = 0.005, respectively). The 8-week RT was also effective in promoting VL µ adaptations (pre= 4.87 ±1.38 kPa and post= 9.08.12 ±1.86 kPa, p = 0.0105), but not in significantly affecting PT µ (pre= 78.85 ±7.37 kPa and post= 66.41 ±7.25 kPa, p = 0.1287) nor PTT (baseline= 0.364 ±0.053 cm and post = 0.368 ±0.046 cm, p = 0.71). The present study showed that an 8-week resistance training protocol was effective in adapting VL µbut not PT µ.Further investigation should be conducted with special attention to longer interventions, to possible PT differential individual responsiveness and to the muscle-tendon resting state tension environment.</div></div>

[STEPS]
TITLE: P7b.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
AUTHORS: cgarcia

[DESCRIPTION]
Atendiendo a las particularidades de la Aplicación LAUREA ACADEMIC, el proceso de presentación del Plan de Investigación inicial por parte de los doctorandos, actualmente queda únicamente bajo la supervisión de tutores y directores. 

Desde la Escuela de Doctorado, creemos que este trámite es fundamental para el desarrollo del Programa y por este motivo facilitaremos a las Comisiones Académicas los mecanismos de control que aseguren que este proceso se realiza correctamente.


[STEPS]
SECTION: P7b.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
1. DESCRIPCIÓN DEL PROCESO

En la primera semana de cada mes, la Secretaría de la EIDUCAM enviará un informe de estado de inscripciones de Planes de Investigación a las Comisiones Académicas. En este informe incluirá la siguiente información:

ALUMNOS PENDIENTES DE INSCRIBIR PLAN DE INVESTIGACIÓN
DNI, NOMBRE Y APELLIDOS, TUTOR, DIRECTOR, ESTADO 

ALUMNOS QUE HAN REALIZADO CORRECTAMENTE LA INSCRIPCIÓN DEL PLAN DE INVESTIGACIÓN
DNI, NOMBRE Y APELLIDOS, TUTOR, DIRECTOR, FECHA APROBACIÓN. OBSERVACIONES

Se considerará inscrito el Plan cuando la aprobación se haya realizado por parte del tutor o cualquiera de sus directores, se hará constar en observaciones quién ha realizado la aprobación y quién está pendiente de realizarla.
SECTION: P7b.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
2. Mensualmente, las Comisiones deberán remitir el informe recibido, resolviendo la aprobación definitiva de los planes de investigación o realizando las observaciones necesarias para que sean trasladadas a alumnos, tutor y director/es. Los informes de resolución de planes deben ser entregados a la Secretaría de la EIDUCAM, antes de la finalización del mes en curso.
SECTION: P7b.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
3. La Secretaría de la EIDUCAM, procederá al registro de planes aprobados.
TITLE: Vu, M-A. T. et al (2024) Targeted micro-fiber arrays for measuring and manipulating localized multi-scale neural dynamics over large, deep brain volumes during behavior
AUTHORS: Mai-Anh Vu

[DESCRIPTION]
This collection contains eight protocols detailing methods used in Vu, M-A. T. et al (2024) Targeted micro-fiber arrays for measuring and manipulating localized multi-scale neural dynamics over large, deep brain volumes during behavior.

[STEPS]
TITLE: Organoids dehydration and Organoids embedding in paraffin
AUTHORS: Gabriela Vallejo Flores, Annika Fendler

[DESCRIPTION]
This protocol is for Organoids dehydration and Organoids embedding adapted from Fendler, A., et al. Research Square (2020).

[BEFORE_START]

Melt the paraffin at 65 °C during 3-6 hrs in the owen.
Fill the Glass bucket with the solutions on step 1.
During the first parafifn incubation time turn on the cooling plate.

[GUIDELINES]
Before starting the protocol,  prepare the follwing solutions:
80% EtOH
90% EtOH
96% EtOH


[STEPS]
SECTION: Dehydration
1. Table 1. Dehydration steps of organoids in agarose beads

 

SECTION: Embedding
6. 
 
    
SECTION: Embedding
2.  With a forceps, carefully transfer the agarose block into a paraffin embedding jar
(metal)

SECTION: Embedding
4. Transfer the embedded jar to a cooling plate, and place the plastic embedding cassette on top on the embedding jar (metal), Incubate for 1h to get solid.
SECTION: Embedding
5. Specimen are now ready for microtom sectioning with subsequent Immunostaining.
SECTION: Embedding
3. Carefully add molten Paraffin (65°C), let stand for at least 2 min at 65°C to ensure
penetration of paraffin into the agarose block 
7.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the untargeted polar analysis of urine samples from cows and calves, with special emphasis on the coverage of thyreostats. Extraction started with 3 mL urine and was in essence based on liquid-liquid extraction with ethyl acetate. Analysis of extracts was achieved by ultra-high performance liquid chromatography (UHPLC) using a Waters Acquity HSS T3 column (1.8 μm, 2.1 x 100 mm), HESI-II ionization, and Q-Exactive Orbitrap mass spectrometry.</p>

[BEFORE_START]
<p>Frozen urine samples were thawed at 4 °C before extraction could be started.</p>
<p> </p>

[GUIDELINES]
<p>Collected urine samples were treated with EDTA (final concentration of 0.1 M) and 0.1 M hydrogen chloride (final pH of 1) to inhibit thyreostat degradation during storage (-20 °C).</p>
<p>The phosphate buffer was adjusted to a pH of 7.</p>

[STEPS]
?. 
?. 
?. 
?. 
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TITLE: Clinical CBD Gummies: Reviews, Ingredients, Shark Tank,  Pain &amp; Is It Scam Or Trusted?
AUTHORS: kairee.jeriko  

[DESCRIPTION]
Why is the discomfort much more astonishing is that often while bodily ache happens, them eats the whole consideration, after which it reminiscence, concentrate, plus all the other mind talents start to reduce and be disabled. Considerable bodily discomfort arise every time they result in melanoma nodules as well as lethal cuboid bone tumours and

>>> Click Here To Visit Clinical CBD Gummies “OFFICIAL WEBSITE>>>

[STEPS]
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Ti (III) Reagent 
AUTHORS: Steven Wilhelm

[DESCRIPTION]
<p>Used for washing off extracellular iron.</p>
<p> </p>
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for any additional information regarding this protocol.</p>

[STEPS]
?. [Chelator Solution]
Per 250 mL, add 4.65 g of Na2EDTA into distilled or Milli-Q water.
[Na2EDTA]
?. [Chelator Solution]
Add 3.68 g of Na3 Citrate.
[Na3 Citrate]
?. [Salts]
Add 2.5 mL of 0.1 M KCl.
[of 0.1 M KCl]
?. [Salts]
Add 3.0 g of NaCl (s).
[NaCl]
?. [Salts]
Add 7.77 mL of 20% TiCl3 while stirring after salts and chelator solution are combined in glass-stoppered bottle.
[20% TiCl3]
?. [Adjust pH]
Using 10 M NaOH, adjust pH to 8. Add NaOH dropwise while stirring to attain desired pH, let equilibrate 30-60 minutes and re-check pH.
[equilibration]
?. [Preparing the Ti(III) Reagent]
To prepare reagent, combine chelator solution and salts in a glass-stoppered bottle. This will be the step where the 20% TiCl3 is added in while stirring.
The final result should be a clear purple solution.
TITLE: Immunofluorescence labelling and imaging of cholinergic interneurons in post-mortem human brain tissues
AUTHORS: Alan K. L. Liu, Laura Parkkinen

[DESCRIPTION]
This protocol details immunofluorescence labelling and imaging of cholinergic interneurons along with striatal astrocytes in formalin-fixed paraffin-embedded (FFPE) post-mortem human brain tissues. 

[STEPS]
SECTION: Collection and fixation of post-mortem human brain tissues
1. Collect 6 µm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues containing the anterior basal ganglia at the level of nucleus accumbens.
SECTION: Collection and fixation of post-mortem human brain tissues
2. Place tissue sections in an oven at 70°C for 30 mins.
SECTION: Collection and fixation of post-mortem human brain tissues
3. Dewax tissue sections in xylene (3 x 5 mins).
SECTION: Collection and fixation of post-mortem human brain tissues
4. Rehydrate tissue sections through decreasing concentration of industrial denatured alcohol (IDA) (100%, 100%, 90%, 70%; 5 mins each)and subsequently in distilled water (5 mins).
SECTION: Primary Antibody
5. Perform heat-induced epitope retrieval by placing tissue sections in autoclave (121°C, 20 mins) in 0.01 M sodium citrate buffer (pH 6.0).
SECTION: Primary Antibody
6. Rinse in PBS (3 x 5 mins).
SECTION: Primary Antibody
7. Incubate samples at 4°C overnight with a mixture of the following primary antibodies: 
anti-ChAT antibodies (1:50, AB144P; Millipore, UK; RRID:AB_2313845)
anti-GFAP antibodies (1:2000; Z0334; Agilent Dako, Santa Clara, United States; RRID:AB_10013382).

 

SECTION: Secondary Antibody
8. Rinse samples in PBS (3 x 5 mins). 
SECTION: Secondary Antibody
9. Prepare Secondary Antibody mixture of Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:200; A-11055; ThermoFisher Scientific, UK) and Alexa Fluor 594-conjugated donkey anti-goat secondary antibody (1:200; ThermoFisher Scientific, UK) diluted in 0.1% Triton-X in PBS.
SECTION: Mounting
11. Incubate sections with TrueBlack® Lipofuscin Autofluorescence Quencher (1:20 with 70% ethanol; Biotium, Fremont, CA, United States) for 30 seconds to block endogenous fluorescence signal.
SECTION: Mounting
12. Rinse in PBS (3 x 2 mins).
SECTION: Mounting
13. Mount and coverslip with Vectashield antifade mounting medium with DAPI (H1900, Vector Laboratories, Peterborough, UK). 
SECTION: Confocal Imaging and Analysis
14. Visualise immunofluorescent-stained slides using Zeiss 880 Airyscan inverted confocal microscope (Carl Zeiss). 
SECTION: Confocal Imaging and Analysis
15. Acquire images using 10x objectives (Plan-Apochromat/0.45 NA) and 63x objectives (Plan-Apochromat, oil immersion; DIC M27/1.4 NA) with laser excitation at 405 nm (solid state), 488 nm (Argon), and 561 nm (solid state).
SECTION: Confocal Imaging and Analysis
16. Perform image capture and processing using the Zen Black and Zen Blue (Carl Zeiss,Germany) software. 
SECTION: Secondary Antibody
10. Incubate sections in Secondary Antibody mixture in the dark (covered with foil) for 1 hour at room temperature.
TITLE: My test protocol
AUTHORS: Abby Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an illegitimate protocol used for testing purposes.</div></div>

[STEPS]
?. [substance combination]
These are my instructions on how to combine substances to make media. Here, I made a tag for NGMA  . I can specify this cat no because it will always be the same.I can also include a field to fill out the substance type, but I would prefer to set a default value for this key instead of having to fill it out via a protocol run.Here, I don't specify the cat no for TB and glycerol because it might change. Also, I don't create a custom key-value set for each component to capture info like mfg, cat no, lot etc. because I don't know how many different cat no and lots I'll have.
[Terrific broth]
[Glycerol]
?. [autoclaving]
These are my instructions on how to autoclave the media from step 1. I can specify a value for duration, but I can't set default values for cycle type and cycle number. I'll have to specify them during the run.Look, protocols.io lets me select an autoclave.
?. [centrifugation]
Here, I'll centrifuge my media. I can specify all my default values with the default centrifugation component in protocols.io.
Centrifuge: 1800 33, 3 min, 20 10
?. [substance combination]
Here, I'm going to make . That tag already existed in protocols.io. I don't know ahead of time the cat no of the bleach I'll use, so I can't specify that with a tag. Here, I'll specify the % v of each component.I can't create a default value for total volume, so I'll include a field here to fill out during my protocol run.
[bleach]
[H2O]
?. [establishing growth environment]
Now I'm going to use   from steps 1-3 to pour into  .I can't specify a default value for the total volume of media poured in the container, but I can create a custom key-value pair. Because I have two different materials listed here, I need to specify what material the total volume applies to.
?. [substance combination]
Finally, I'll make a solvent. I know ahead of time what the mfg and cat no will be for my components.
[Methanol]
[H2O]
?. [seed]
Let's seed the growth environment made in step 5 using the   made in step 6.There's no default component for me to specify the number of organisms to seed, so I'll create a custom field to specify that during the run.
TITLE: Zeiss AxioImager 63X Z-Stack Image Capture
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the system that is used to capture Z stack images which are reassembled post-processing into 3D reconstructions.

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: Overlap PCR
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to combine DNA fragments with overlapping regions, thereby reducing the number of fragments used in the Gibson Assembly and thus, improving the success rate of the Gibson Assembly. These overlapping regions are created during the PCR procedures by designing forward and reverse primers for each fragment to contain overlapping sequences.

[GUIDELINES]
This protocol outlines a general method for combining two DNA fragments with overlapping regions through overlap PCR. The number of fragments to be overlapped can be increased by adjusting the volume ratio between each DNA fragment.

[STEPS]
1. Determine the volume ratio for each DNA fragment required in the reaction to calculate the appropriate volume for each DNA fragment. The final sample volume is 47 µL, with 25 µL being the KOD OneTM PCR Master Mix (Blue). Therefore, the remaining volume (22 µL) will be allocated to the gene fragments and DI Water for topping it up. 

*This calculation requires the fragment size and the concentration of the target DNA fragments.
1.1. Divide the bigger fragment size by the smaller fragment size to get the volume ratio of the DNA fragment with the bigger fragment size.                      
1.2. Divide the concentration of the bigger fragment by the answer obtained in the previous step.  
1.3. Divide the number obtained by the concentration of the smaller fragment to get the volume ratio of the DNA fragment with the smaller fragment size.                      
1.4. The volume ratio of each fragment may be adjusted to reach the final sample volume of 47 µL (DI water will be used to top up the remaining volume).

2. Make the 47 µL PCR sample by adding the following into a PCR tube:

 

3. Place the PCR tube into the Thermal Cycler and set the conditions to:

 

5. After adding the primers, cancel the previous run protocol immediately and reset the conditions of the Thermal Cycler to:

 

4. In Step 5 of the above condition, the "Extension" step, quickly add 1.5 µL of each primer into the PCR tube before the 5-minute duration is completed.
 

6. Proceeds to the gel electrophoresis to isolate the combined gene fragment.
TITLE: Prescholers&#39; causal reasoning
AUTHORS: Tessa Van Schijndel, Kim Huijpen, Ingmar Visser, Maartje Raijmakers

[DESCRIPTION]
1 Protocol
2-5 Scoring forms (versions 1A, 1B, 2A and 2B)
 
For communication regarding the translation of the documents the authors can be contacted.

[STEPS]
TITLE: Novel Object Recognition Test
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Novel Object Recognition Test for mice

[STEPS]
1. Individualize each mouse in a different cage with new bedding. 
2. Expose the animal to two identical objects for habituation.
3. After 24h change one of the two objects for a new one.
4. Measure the time the animal spends exploring the new object and the old
object. 

5. Calculate the discrimination index using the formula: [time exploring the
new object-time exploring the familiar object]/total time).
TITLE: Total Cellular RNA Purification Protocol from Animal Tissue (Trizol + RNeasy)
AUTHORS: Loyal Goff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides an optimaized, high-quality total RNA extraction method that balances time and RNA-yield effectively by combining the phase separation properties of Trizol with the efficiency of glass fiber filter column binding. Tissue homogenization by mortar & pestle in LN2 ensure minimal RNA degradation and efficient lysis in Trizol.</div></div>

[STEPS]
?. [Sample Homogenization]
Begin with  tissue stored at (for each  TRIzol you will use).
50 mg
-80 °C
1 mL
?. [Sample Homogenization]
Place tissue in liquid nitrogen and grind to a powder with a frozen mortar and pestle.
?. [Sample Homogenization]
Liquid nitrogen will evaporate, but prior to tissue thawing, add TRIzol andvortex to mix well.
1 mL
?. [Sample Homogenization]
Pulse spin to collect contents, and place on ice.
?. [Sample Homogenization]
Decant powder and liquid nitrogen into an RNAse-free frozen microcentrifuge tube  (using a frozen spatula as a guide).
?. [Phase Separation]
Add  of chloroform to each tube and vortex each sample for  (or until cloudy). Place sample on ice for . Centrifuge at  for  at .
100 µl
Centrifuge: 13.000 33
4 °C
?. [Phase Separation]
Remove about  of the upper aqueous phase from each tube and place it in a new tube. While removing sample, be sure to completely avoid the interface and the organic phase. Save the organic phase if isolation of DNA or protein is desired (as per TRIzol package instructions).
250 µl
?. [Phase Separation]
Slowly add  (in  aliquots) of 70 % ethanol at  to tube (equal parts 70% ethanol and aqueous phase). Mix thoroughly after each aliquot.
250 µl
50 µl
25 °C
?. [RNA consolidation]
Apply sample to RNeasy mini-spin column in 2mL collection tube.
?. [RNA consolidation]
Centrifuge at r for  at , and discard flow- through.
25 °C
Centrifuge: 10.000 33
?. [Wash]
Pipette  of RW1 onto the column.
700 µl
?. [Wash]
Centrifuge at   for at .
25 °C
Centrifuge: 10.000 33
?. [Wash]
Discard flow-through and collection tube.
?. [Wash]
Put column in new 2mL collection tube.
?. [Wash]
Pipette  RPE solution ( RPE in  100% ethanol) on column. Centrifuge for  at  and discard flow-through.
500 µl
100 µl
400 µl
Centrifuge: 10.000 33
?. [Wash]
Pipette  RPE solution (RPE in  100% ethanol) on column Centrifuge for  at maximum speed.
500 µl
100 µl
400 µl
?. [Elution]
Transfer RNeasy column into new 1.5mL collection tube.
?. [Elution]
Slowly pipette  pre-warmed () RNase-free water onto the center of the RNeasy membrane.
50 µl
50 °C
?. [Elution]
Centrifuge for  at
Centrifuge: 10.000 33
?. [Elution]
Repeat steps 18 and 19 as needed to fully elute RNA from column.
?. [Elution]
Wait  (incubation needed only if yields are low).
?. [Elution]
Once off column, place eluate (RNA) on ice.  Store at .
-80 °C