eamag/protocols-with-steps huggingface dataset:
TITLE: SPOT1 assay
AUTHORS: Guanhua Xun, Huimin Zhao, stlane2 

[STEPS]
?. Using the first provided microcap, a sample is collected into capillary A, containing QuickExtract DNA Extraction Solution (Lucigen).  Insert the capillary into the SPOT1 device and press the "Start" button to run the 5-minute pretreatment.
95 °C
?. After pretreatment, remove capillary A from the SPOT1 device and use the second provided microcap to transfer a small volume of pretreated sample to capillary B, which contains the SPOT assay mastermix.  Insert capillary B into the SPOT1 device and press the "Start" button to initiate the 35-minute detection reaction.SPOT assay mastermix:  ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
63 °C
98 °C
ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
2 µl
1 µl
8 µl
5.6 µl
3.2 µl
0.8 µl
?. Result ("Positive"/"Negative"/"Inconclusive") will be displayed on SPOT1 device LCD screen after completion of detection reaction and the 1-minute cooling period.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The second half of the workshop was an exercise that I copied from Professor Elizabeth Sargent.</p>
<p>https://twitter.com/esargent184/status/1009430882413883394</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/4tacz36.png" data-src="https://s3.amazonaws.com/pr-journal/4s9cz36.png" data-ofn="Screen Shot 2018-07-31 at 11.56.47 AM.png" /></p>
<p> </p>
<p>Results from our class are here:</p>
<p><a href="https://www.protocols.io/groups/fsci/protocols" target="_blank" rel="noopener noreferrer">https://www.protocols.io/groups/fsci/protocols</a></p>

[STEPS]
?.
TITLE: Fecobionics Test in Subjects with Fecal Incontinence
AUTHORS: Yanmin Wang, Hans Gregersen

[DESCRIPTION]
It is the protocol of Fecobionics test in normal subjects. Briefly, the device will be inserted into participants' rectum and several motions (cough, squeeze, straining) will be done. After the motions, the bag will be inflated, and the participant may have different sensations during inflation. If the device falls out before the completion of the test, it will be re-inserted gently. Data will be recorded and shown in real time.
Each participant will be tested up to 4 times at following dates: The day before percutaneous nerve evaluation (PNE) (pre-PNE), one day after PNE (day 1), seven days after PNE (day 7), and thirty days after sacral nerve stimulation (SNS) treatment (day 30 post-SNS).

[STEPS]
1. Make sure the participant is diagnosed as fecal incontinence and eligible to do the study. Make sure he/she understands the study and has signed the informed consent form.
2. Ask the subject to empty bladder and rectum before the test.
3. Participant changes clothes and lies on left side.
4. Calibrate the Fecobionics device and make sure the transmission signal is good.
5. Fully lubricate Fecobionics device. Insert device gently into rectum, following a finger examination.
6. Participant moves to commode, in sitting position. Wait up to 5 mins until pressure data is stable.
7. Ask the participant to cough twice, with 15-30 seconds in between.
8. Ask the participant to squeeze three times, 5 seconds for each time, and 15-30 seconds in between. 
9. Ask the participant to do a long squeeze that lasts 30 seconds.
10. Ask the participant to do a straining (contract both abdominal and anal muscles) that lasts 20 seconds.
11. Inflate the balloon with warm saline. Ask the participant to report sensations.
12. Stop inflating when the participant feels urge, or the maximum volume (100ml) is reached.
13. Detach the filling tube and give the participant privacy to defecate. If the participant cannot defecate Fecobionics in 3 minutes, withdraw it manually by pulling the thread attached to the device.
14. Test complete. Save data and clean up.
TITLE: ViaKeto BHB Apple Gummies Reviews! 
AUTHORS: ViaKeto BHB Apple Gummies

[DESCRIPTION]
CHOOSE YOUR COUNTRY FOR ORDER VIAKETO GUMMIES!

| ORDER VIAKETO APPLE GUMMIES IN UNITED KINGDOM |

| ORDER VIAKETO APPLE GUMMIES IN UNITED STATE |

| ORDER VIAKETO APPLE GUMMIES IN FRANCE |

| ORDER VIAKETO APPLE GUMMIES IN CANADA |

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> After total genomic DNA was extracted, complete cytochrome <em>b</em> (cyt <em>b</em>) gene and 11 microsatellite loci were examined for all sampled individuals. Based on the cyt <em>b </em>sequences, phylogenetic analyses with maximum likelihood and Bayesian inference methods were conducted and a median-joining haplotype network was calculated.The divergence time between major lineages was estimated using BEAST. Genetic structure based on the microsatellite dataset were also analyzed to detected admixture between major lineages in the mixed region.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans
AUTHORS: Coline Royaux, Nicolas Rabet, Céline Bonillo

[DESCRIPTION]
This PCR protocol has been used to amplify the Cytochrome Oxydase I gene of neo-caledonian genera Boeckella, Lynceus, Eulimnadia and Streptocephalus with several primers.

[STEPS]
1. Prepare the mix for the PCR depending on the quantity of DNA samples you want to amplify
 

 



1.1. For each well, mix 12.44 µL, 2 µL, 1 µL, 1 µL, 0.8 µL and 0.12 µL. This mix is hereafter named "intermediary mix".
 


1.2. Add your primers to the mix, 0.32 µL 10 picomolar (pM) and 0.32 µL 10 picomolar (pM)  for each well. This mix is hereafter named "final reagent mix".
 

 



2. In each well, deposit 18 µL and 2 µL unless for the negative control.
For the positive control, use the DNA of the species on which the primer has been designed.

3. Close the PCR plate with a heated aluminium foil and centrifuge it enough to make sure the reagents are all in the bottom of the well.
4. To amplify targeted DNA sequences, place your plate in the thermocycler, set and launch the cycle as described in the following sub-steps
4.1. 5 min at 94 °C 

4.2. 40 cycles of the following steps :
30 s at 94 °C 
30 s at 50 °C 
1 min at 72 °C 

4.3. 8 min at 72 °C 

4.4. Infinite timing at 20 °C so the samples are well preserved and you have some time to pick it up

5. To know if your amplifications worked properly, you now have to proceed to a gel electrophoresis of your PCR products
5.1. To prepare the gel,
 

 mix 0.8 mg and 40 mL, heat the preparation in the microwave for a few moments until it is homogeneous (you can agitate the preparation). Wait until the preparation is cold enough to touch its container and add 0.8 µL.
5.2. Pour the preparation in a mold and let the gel polymerize with a bar to form wells to deposit your PCR products
5.3. Deposit the gel in the electrophoresis tank
5.4. Fill each well with 2.5 µL and 0.5 µL , don't forget to save a well to deposit 1.2 µL 

5.5. Start electrophoresis in TAE buffer for 15 min at 200 Volts

5.6. Look at your gel under Ultra Violet light to see the results
TITLE: Reverse transcription, primer pools preparation and multiplex PCR steps for CHIKV serotype  genomic sequencing 
AUTHORS: Laís Ceschini, Luisa Maria Inácio da Silva, Gabriel Luz Wallau

[DESCRIPTION]
This step-by-step protocol describes the cDNA synthesis, primer pools preparation and multiplex PCR conditions with the main goal to sequence the complete genome of CHIKV serotype strains.

[STEPS]
SECTION: Reverse transcription
1. Using a 2mL tube prepare the Mix 1 described below for 96 samples:

 


SECTION: Reverse transcription
2. Using 0,2mL PCR tubes or 96 wells plates add 11-16µL of extracted RNA from
RT-PCR positive samples. Add 2µL of Mix 1 to the tube/well and take it to the
thermocycler with the following set up:

65ºC ---- 5 minutes

SECTION: Reverse transcription
3. Take the tubes/wells to ice for 1 minute. (you can prepare a water bath with ice cubes to have a uniform temperature distribution).

SECTION: Reverse transcription
4. Using a 2mL tube prepare Mix 2:
 

SECTION: Reverse transcription
5.  Add 7µL of Mix 2 to the tubes containing the Mix 1 plus RNA and take it to the thermocycler following the set up below:

Step1:
42ºC ---- 50 minutes
70ºC ---- 10 minutes
4ºC ---- Hold

SECTION: Reverse transcription
6. Store the cDNA at -20ºC.

Observation:. As a suggestion, to improve the final results only samples RT-PCR positive showing a Ct value of < 30 should be used for cDNA conversion and genomic amplification.
SECTION: Pools of primers
7. Select two 0,6mL tubes for each pool.
SECTION: Pools of primers
8. Using the original 100uM primer solution eluted individually, put them together following the table below containing each primer volume.
SECTION: Pools of primers
9. Pool 1 will have a final volume of 469µl and pool 2 of 460µl.
SECTION: Pools of primers
10. In order to prepare the solution to use in the Multiplex PCR, dilute each pool 1:10. That is, 10µl of pool 1 and 90µl of ultrapure water.

TABLE 1: Primers and pool order


 
*approximate concentration of each primer in the 25µl PCR reaction.

Note: The primers were designed using the https://primalscheme.com based on the
KP164576, KP164571, KP164572, KP164568, KP164570 and KP164569 reference genomes (Machado, 2019).

SECTION: Multiplex PCR
11. Prepare the Mix 1 for a Multiplex PCR for each Pool 1 and Pool 2 using a Falcon tube
of 15mL (~96 amostras) or a 2mL tube.


 

SECTION: Multiplex PCR
12. Add 2,5µl of cDNA (totalling 5µl) in 22,5µl of the pool1 and pool2 reaction and take
it to the thermocycler following the conditions bellow:

Step1:
98ºC ---- 30 seconds
Step2: (45 cycles)
98ºC ---- 15 seconds
58ºC ---- 30 seconds
72ºC ---- 5 minutes
Step3:
72ºC ---- 2 minutes
Hold 4ºC
TITLE: Enhancing precision flood mapping: Pahang&#39;s vulnerability unveiled
AUTHORS: Dr. Tahmina Afrose Keya, Siventhiran S B, Maheswaran S, Sreeramanan S, Low J An, Leela A, Prahankumar R, Lokeshmaran A, Boratne AV, Abdullah, M. T

[DESCRIPTION]
Flooding in Malaysia is considered one of the most impactful natural disasters. Annually, Pahang experiences substantial destruction due to floods. The aim of this research is to address the urgent issue of flood susceptibility in Pahang, Malaysia. To achieve this, a combination of Geographic Information System (GIS) and Ensemble Machine Learning (EML) will be utilized. By considering nine factors from a geospatial database that contribute to flooding, the areas prone to floods will be mapped. The mapping process will be carried out using the ArcGIS environment, and a model called Random Forest (RF)-embedding will be developed using the Ensemble Machine Learning (EML) technique. To determine the most influential factors in flooding, Feature Selection (FS) will be employed. The accuracy of the flood susceptibility models will be assessed by analysing the Area Under the Curve (AUC). Flood susceptibility mapping is a complex procedure with uncertainties. Hence, our research can contribute to flood management in vulnerable regions by improving flood models and providing spatial outcomes to help decision-makers implement risk reduction strategies.

[BEFORE_START]
GIS is essential for spatial data analysis and decision-making, particularly in flood susceptibility mapping. It integrates geospatial data to examine spatial relationships and visualize vulnerable areas. Machine Learning, specifically ensemble methods like Random Forest, provide advanced techniques for analysing complex datasets and improving the accuracy of flood susceptibility predictions.

[STEPS]
SECTION: Conceptual framework
1. Develop an Integrated GIS-Based Framework. 
The objective is to establish a robust GIS-based framework for flood susceptibility mapping in the Pahang
State. This involves compiling and integrating geospatial datasets related to topography, hydrology, land use, and climate variables to create a comprehensive database for analysis.
SECTION: Conceptual framework
2. Apply Ensemble Machine Learning Algorithms. 
The objective is to apply ensemble machine learning algorithms, such as Random Forest (RF) and Gradient Boosting Machines (GBM), to the integrated dataset to develop predictive models of flood susceptibility.     
SECTION: Conceptual framework
2.1. This objective includes feature selection, model training, validation, and evaluation to ensure the accuracy and reliability of the susceptibility maps.
SECTION: Conceptual framework
3. Generate Actionable Insights for Decision-Making: 
The objective is to generate actionable insights from the flood susceptibility maps to support informed decision-making and disaster management strategies. This involves identifying vulnerable areas, assessing the factors contributing to flood risk, and recommending targeted interventions and mitigation measures to reduce the impacts of floods on communities, infrastructure, and the environment.
TITLE: The ARF-AID system: Methods that preserve endogenous protein levels and facilitate rapidly inducible protein degradation
AUTHORS: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The ARF-AID (Auxin Response Factor-Auxin Inducible Degron) system is a reengineered auxin-inducible protein degradation system. Inducible degron systems are widely used to specifically and rapidly deplete proteins of interest in cell lines and organisms. An advantage of inducible degradation is that the biological system under study remains intact and functional until perturbation. This feature necessitates that the endogenous levels of the protein are maintained. However, endogenous tagging of genes with AID can result in chronic, auxin-independent proteasome-mediated degradation. The additional expression of the ARF-PB1 domain in the re-engineered ARF-AID system prevents chronic degradation of AID-tagged proteins while preserving rapid degradation of tagged proteins. Here we describe the protocol for engineering human cell lines to implement the ARF-AID system for specific and inducible protein degradation. These methods are adaptable and can be extended from cell lines to organisms. </div></div>

[STEPS]
TITLE: FireLAMP Protocol
AUTHORS: Daniel Benner, Ozlem Yaren

[STEPS]
?. SETTING UP THE GENIE II
?. Take a swab
?. Preparing a sample batch (up to 8 samples different testees)
?. Plug In
?. Set the block temperature to. Make sure temperatures are reached to set temperature before placing the assay tubes into heat-block for amplification.
65 °C
?. Open sample swab with caution and transfer swab to a blue tube, which contains elution solution and mix/swirl the swab 4-5 times.
?. Pipette  of eluted swab sample from the blue tube into a tube on the red strip, which contains SARS-CoV-2 master mix. Pipette up and down 2 times to mix assay components, then close the lid
5 µl
?. Pipette  of eluted swab sample from blue tube into a tube on the green strip, which contains the internal control master mix. NOTE: samples from each patient should be put in the same tube number on the red strip and green strip
5 µl
?. Hand testee a sterile swab, packaged in 15 mL falcon tube, and instruct patient on use
?. Testee to remove swab from 15 mL falcon tube
?. Testee returns swab to 15 mL falcon tube
?. Testee inserts swab into each nasal cavity, rotating for 10 seconds
?. Testee hands tube to supervisor
?. Discard the swab in a biohazardous waste container
?. Repeat steps 4.1 through 4.4 for all samples in the batch
?. Running a sample
?. On the Genie II touch screen, select blocks A and B
?. Run program called "FireLAMP"
?. Wait for incubation to complete (~30 minutes for negative samples)
?. Preparing for Next Block
?. Discard all used blue tubes, red strips, and green strips in biohazard container
?. Wait 5 minutes for Genie II to cool before starting analyis of a new batch.
?. Setting up Workplace with Reagents
?. Take Red Strips and Geen Strips out of freezer and thaw for 1-2 min at room temperature
?. Set up biohazard disposal
?. Set Blue Tubes in work area
?. Open the . Place the red batch into Block A and the green batch into Block B. Close the Genie II.
?. Interpretation of results
?. Touch results tab.
?. Record results based on the "results column" column for block A and block B. Positive result in the block with the red strip (typically block A) indicate a positive result for COVID19. Positive results in the block with the green strip (typically block B) indicate that the sample was taken correctly.
?. Record results
TITLE: Investigaciones de impacto que estudian los factores contextuales que afectan a la Eficacia Escolar en el periodo 2014-2019
AUTHORS: Pablo Delgado-Galindo, Javier Rodríguez-Santero, Juan Jesus Torres-Gordillo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">La tabla forma parte de una revisión sistemática de investigaciones de impacto del periodo 2014-2019 que muestran los factores contextuales que influyen en la eficacia escolar. El objetivo de conocer estos factores es tener un mayor conocimiento de qué influye en la eficacia escolar para mejorar las prácticas orientadoras. </div><style>
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							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">La tabla recoge las publicaciones ordenadas de mayor a menor factor de impacto en su año de publicación. En el caso de los libros y capítulos de libro, se indica su inclusión en el ranking Scholary Publishers Indicators (SPI). Las tesis doctorales se encuentran en repositorios institucionales de prestigio. </div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The assay was carried out following the protocol reported by Dineshkumar et al. [8]. Starch (2 mg) was suspended in a tube containing 0.2 mL of 0.5 M Tris-HCl (Sigma-Aldrich, USA) buffer (pH 6.9) with 0.01 M calcium chloride as substrate. The tube was boiled for 5 min and then preincubated at 37 °C for 5 min. Plant aqueous extract (1 mg) was dissolved with 1 mL of 0.1% of dimethyl sulfoxide in order to obtain a concentration of 1,000 µg/mL; then 0.2 mL of aqueous extract was added to the tube containing the substrate solution, 0.1 mL of porcine pancreatic amylase in Tris-HCl buffer (2 U/mL) was also added, and incubated for 10 min at 37 °C. Finally, the reaction was stopped with 0.5 mL of acetic acid (50% v/v) and centrifuged 5 min at 1,811 × g and 4 °C. The assay was performed in triplicate. The a-amylase inhibitory activity was calculated using the formula (Ac<sup>+</sup>) – (Ac<sup>-</sup>) – (As - Ab)/(Ac<sup>+</sup>) – (Ac<sup>-</sup>) × 100, where Ac<sup>+</sup>, Ac<sup>-</sup>, As, Ab are defined as the absorbance (595 nm) of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme), test sample (with enzyme), and a blank (a test sample without enzyme), respectively.</p>

[STEPS]
TITLE: Liposome binding
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for LRRK2-liposome binding experiments analyzed by co-sedimentation or confocal fluorescence microscopy, respectively.

[STEPS]
SECTION: Co-sedimentation analysis
1. Prepare samples in Beckman microfuge tubes with 300 nanomolar (nM) LRRK2 or RCKW, 20 micromolar (µM) PS liposomes in the absence or presence of 1 millimolar (mM) GMPPNP.
SECTION: Co-sedimentation analysis
2. Incubate samples at 37 °C for 30 min.
SECTION: Co-sedimentation analysis
3. Centrifuge samples at 49000 rpm (100000 x g) for 20 min in a Beckman Optima TLX ultracentrifuge.
SECTION: Co-sedimentation analysis
4. Collect Supernatants and Pellets.
 

SECTION: Co-sedimentation analysis
5. Analyze samples by SDS-PAGE and coomassie blue staining.
SECTION: Confocal fluorescence microscopy analysis
6. For PS liposome binding. Prepare samples in PCR tubes with 20 micromolar (µM) Rhod-PE labeled PS liposomes and different concentrations of LRRK2 as indicated in the main text.
SECTION: Confocal fluorescence microscopy analysis
7. Immediately deposit 6 µL-10 µL samples of step 6 on   glass bottom dishes and incubate at 37 °C for 30 min.
 

SECTION: Confocal fluorescence microscopy analysis
8. After incubation, capture the images with a spinning disk confocal (SDC) microscopy at Room temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system (Perkin-Elmer).
SECTION: Confocal fluorescence microscopy analysis
6.1. For GC/PS nanotube binding. Prepare samples in PCR tubes with 20 micromolar (µM) Cy5-PE labeled GC/PS nanotubes and 100 nanomolar (nM) GFP-LRRK2.