eamag/protocols-with-steps huggingface dataset:
TITLE: TSS, TXS, SDS Serial Extraction
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to serially extract protein from mouse brain tissue in increasingly stringent buffers.

[STEPS]
1. Weigh brain sample and add 3X volume of TSS buffer + 1X protease inhibitor.
TSS Buffer: 140mM NaCl + 5mM Tris-HCl in H2O
2. Homogenize in a dounce homogenizer then transfer to a clean tube.
3. Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TSS fraction'.
4. Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
5. Resuspend the pellet in the same volume of TXS buffer then incubate on ice for 10 minutes.
TXS Buffer: TSS buffer + 0.5% Triton X-100
6. Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TXS fraction'.
7. Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
8. Resuspend the pellet in the same volume of SDS buffer then incubate at room temperature for 10 minutes.
SDS Buffer: TSS buffer + 1% sodium dodecyl sulfate (SDS)
9. Centrifuge at 21130g for 30 minutes at room temperature then save the supernatant as 'SDS fraction'.
TITLE: Orthogonal experiment of research on coordinated control method of indoor multiple pollutants
AUTHORS: Yan Zhang, Wei  Yu

[DESCRIPTION]
Indoor air pollution in office buildings is complex and serious. This research is targeted at three types of air pollutants: CO2, VOCs, and PM2.5. In order to seek the optimal control method to achieve good indoor air quality, an orthogonal experiment was carried out in the environmental chamber to compare the control time and energy consumption of four control methods (purifier+ and window+, purifier+ and window-, purified fresh air 240 m3/h and purified fresh air 400 m3/h) required for pollutants to meet the standard. The result shows that the purifier+ and window+ method is more effective in most conditions. Its control time is reduced by 8.06% and the energy consumption is reduced by 11.91% compared with the traditional control method of purified fresh air 240 m3/h. This research guides the optimization control strategy of indoor air quality under different pollution conditions in office buildings.

[BEFORE_START]
Calibrate the instrument. Close the doors and windows. Switch on all temperature, humidity, and IAQ sensors. Switch on the air purification equipment until the concentrations of CO2, VOCs, and PM2.5 are reduced to the lowest level. Then switch off the purifiers.


[GUIDELINES]
This experiment aims to obtain the optimal coordinated control method under different multiple pollutants conditions; provide guidance for building designers, building occupants, and building managers on the comprehensive management of indoor multi-pollutants for excellent indoor air quality.

[STEPS]
1. Turn on the air conditioning. Turn on all the temperature, humidity, and indoor air quality sensors. Close the doors and windows. Start the experiment.
2. Create the indoor air pollution sources artificially until the CO2, VOCs, and PM2.5 in the breathing zone have exceeded the values set for each condition, and then stabilize for 2 minutes.
3. Turn on the air purifier or purified fresh air system according to the condition, and set the fresh air volume. Turn on the time monitor and energy power monitor to record the control time and energy consumption.
4. When all the IAQ sensors show that CO2, VOCs, and PM2.5 are below the standard, record the time and energy power again. Then stabilize for 5 minutes.
5. Turn off all the equipment and sensors and end the experiment.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 1">
<div class="section">
<div class="layoutArea">
<div class="column">Estimate a protein concentration in a solution using the Bradford protein assay</div>
</div>
</div>
</div>

[STEPS]
?. 
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?. 
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?. 
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?.
TITLE: A First Experimental Protocol on Creative Engagement and Meaning Creation in Interactive Experiences for Cultural Heritage 
AUTHORS: Manuele Veggi, sofia.pescarin

[DESCRIPTION]
The following document contains the descriptive material for the testing protocol of the "caring prototype" MyTISSE, an interactive user experience on Matisse's painting Bathers by a River (Art Institute of Chicago). Focusing on the change of the pigments made by the artist during the gestation of this canvas, the UX exploits creative engagement to catalyze meaning creation processes.

The attachments contain:
the Informed Consent Form 
the outline of the presentation of the painting
the transcription of the form (handed to participants via Google Form)

[STEPS]
SECTION: Preparatory Activities
1. Preparation of the material:
Tablet interface (XRF scan and two palettes)
Informed Consent form
Outline of the analysis of the painting
Digital survey (with QR code to access it)
SECTION: Creative Engagement
5. Participants are given a tablet and an interactive pen

 

SECTION: Creative Engagement
6. Instructions are given to the participants: identify human figures, parts of them and other elements and color them using only one of the two palettes 
SECTION: Creative Engagement
6.1. Group A Participants are told that the XRF refers to a painting is by Matisse. No other piece of information is provided. They are also asked to provide a title
SECTION: Creative Engagement
6.2. Group B Participants are told that the XRF refers to the painting "Bathers by a River" by Matisse 
SECTION: Creative Engagement
6.3. Group C Participants are told that the XRF refers to the painting "Soldiers by a River" by Matisse 
SECTION: Creative Engagement
7. Participants choose only one of the available two palettes
SECTION: Creative Engagement
8. Participants freely draw and color the XRF image

 

SECTION: Knowledge Acquisition
9. Participants are provided a small presentation of the canvas and of its gestation

 


SECTION: Evaluation
10. Participants scan a QR Code

 

SECTION: Evaluation
11. Participants fill the survey

 


 

SECTION: Arrival of the participants
3. Participants are given an alphanumeric identifier
This ID is composed of one letter, referring to the group, and one number, referring to the participant within the group
5. 
SECTION: Arrival of the participants
4. Participants fill in the Informed Consent form

 

4. Pfill in the Informed Consent form
SECTION: Preparatory Activities
2. Participants are divided in three groups and distributed in the available time slots
TITLE: General Transfection
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for general transfection. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/transfection/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bkyxe.png" /></div></div>

[STEPS]
?. [Seeding cells]
Seed 293T packaging cells at 3.8x106 cells per plate in DMEM complete in 10 cm tissue culture plates.
?. [Seeding cells]
Incubate the cells at , 5% CO2 for ~.
37 °C
?. [Transfection]
Gently aspirate media, add  fresh DMEM complete containing cloroquine diphosphate and incubate ~.
10 ml
For   of DMEM complete, add   of   chloroquine diphosphate.
?. [Transfection]
Dilute  of DNA into  of Opti-Pro SFM.
18.9 µg
500 µl
*Pro-Tip* Endotoxins can inhibit transfection, therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable.
?. [Transfection]
Dilute 1:3 (ug DNA:ug PEI) in  total of OptiPro SFM (per 10 cm plate).
500 µl
*Pro-Tip*The ratio of ug DNA:ug PEI needs to be empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells.
?. [Transfection]
Step 5 example:  of 1 mg/mL PEI, MW 25,000 Da in  of OptiPro SFM per 10 cm plate.
56.7 µl
386.6 µl
?. [Transfection]
ABC12Ratio of DNA:PEIAmount of DNA (μg)Volume of 1 mg/mL PEI (μL)31:118.918.941:218.937.851:318.956.761:418.975.671:518.994.581:618.9113.4
ABC12Ratio of DNA:PEIAmount of DNA (μg)Volume of 1 mg/mL PEI (μL)31:118.918.941:218.937.851:318.956.761:418.975.671:518.994.581:618.9113.4
?. [Transfection]
Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture  -  at .
0 Room temperature
?. [Transfection]
Gently add the diluted PEI to the diluted DNA.
?. [Transfection]
Add the diluted PEI dropwise while gently flicking the diluted DNA tube.
?. [Transfection]
Incubate the mixture  -  at .
0 Room temperature
?. [Transfection]
Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells.
?. [Transfection]
Incubate the cells for , or until the following morning.
?. [Transfection]
The following morning, carefully aspirate the media. Replace the media with  of DMEM complete.
15 ml
?. [Transfection]
Incubate the cells  -  before checking for protein expression.
TITLE: Tracking bleach synchronized worms
AUTHORS: Priota Islam

[STEPS]
?. [Preparing Imaging plates]
Prepare and pour low peptone NGM onto 35mm imaging plates  Put them in the cold room for at least 2 days before use Seed imaging plates with 50ul of freshly made 1:10 solution (OP50:M9 solution) the day before recording  Leave on bench to dry with lid on overnight
?. [Preparing worms]
Chunk the worms on 3 maintenance plates Bleach the worms after 2 days following the protocol for Bleach synchronization of c elegansRefeed the starved L1s on 2 different NGM plates that are OP50 seeded, 72hr prior to the experiment day (Repeat for every strain to be tracked)
?. [Day of recording]
Using a hair pick – pick 5 young adults onto the seeded imaging plates Put the plates on the tracker – agar side up with the lids off Wait 30min for the worms to acclimatise Record for 15min (See protocol Imaging on the multiworm tracker for detailed tracking instructions)
TITLE: Microbial Drug Discovery
AUTHORS: contact.microbialtec  

[DESCRIPTION]
Drugs of natural origin have been classified as (i) original natural products, (ii) products derived or chemically synthesized from natural products or (iii) synthetic products based on natural product structures. Microbes have made a phenomenal contribution to the health and well-being of people throughout the world. In addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products. For example, immunosuppressants have revolutionized medicine by facilitating organ transplantation. Other applications include antitumor drugs, enzyme inhibitors, gastrointestinal motor stimulator agents, hypocholesterolemic drugs, and other pharmacological activities.

[STEPS]
TITLE: Statistical Code Summary for "Predicting the 10-Year Risk of Death from Other Causes in Men with Localized Prostate Cancer Using Patient-reported Factors: Development of a Tool"
AUTHORS: Daniel Frendl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This text document summarizes the steps used for managing the data and performing the core analyses reproted in the manuscript. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>With the booming development of evacuation simulation software, developing an extensive database in indoor scenarios for evacuation models is imperative. In this paper, we conduct a qualitative and quantitative analysis on the collected videotapes and aim to provide a complete and unitary database of pedestrians' earthquake emergency response behaviors in indoor scenarios, including interactions between man and the environment. Using the qualitative analysis method, we extract keyword groups and keywords that code the response modes of pedestrians in indoor scenarios and construct a general decision flowchart using chronological organization. Using the quantitative analysis method, we analyze data on the delay time, evacuation speed, evacuation route and emergency exit choices. Furthermore, we study the effect of classroom layout on emergency evacuation. The database in indoor scenarios provides reliable input parameters and allows the construction of real and effective constraints for use in software and mathematical models. It can also be used to validate the accuracy of evacuation models.</p>

[STEPS]
?. 
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?. 
?.
TITLE: Structuralization and Incubation
AUTHORS: Laura Armero H, Claudia Troncone Clemente

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The interaction between an aptamer and his target depends on the tertiary structure of the aptamer, to attempt to always have the same structure for all the rounds, we forced the aptamer population to achieve the most thermodynamic stable structure.</div><div class = "text-block">Once all the sequences are structuralized, we can incubate the library with our target.</div></div>

[STEPS]
?. Strcucturslization
?. [Day 1 ]
Strcuturalization
?. [Day 1 ]
Add 10 μL of 100 uM ssDNA library to 190 ul of binding buffer. Incubate it at 95 °C for 10 min. After that, transfer immediately to ice-bath to prevent rehybridization ofthe single-stranded DNA library. If the transfer is poor performed, you can reheat again for another 10 min and repeat.Incubated at 4ºC for 10 min. Then you attempered the library at room temperature for 10 min.
?. [Day 1 ]
Incubation
?. [Day 1 ]
Resuspend 100 μL of 1 x 103 cells and add your library aptamer into a 1.5 ml Eppendorf tube. Place the tube into the thermoblock set and 40 ºC and incubate with agitation for 1h.
TITLE: Splenocyte Preparation for Immune Reconstitution (Humanisation) 
AUTHORS: Sandra Petrus-Reurer, Kourosh Saeb-Parsy

[DESCRIPTION]
This protocol guides through the steps required to humanise mice with human splenocytes

[STEPS]
1. Thaw vial (approx. 50x106 cells/vial) in water bath 37C for 2-3min (leave crystal)
2. Add cells into 10mL/vial of warm 50% RPMI media + 50% FBS 
3. Centrifuge 7min at 300g
4. Combine all vials in 10mL RPMI media + 10% FBS 
5. Centrifuge 7min at 300g
6. Resuspend all vials in 10mL RPMI media + 10% FBS + add DNAse-I (typically 40uL/mL if DNAse-I 1mg/mL) at 37C (incubator) for 15-20min (depending on the amount of clumps seen). Check and flick the tube every 5 min
7. Centrifuge 7min at 300g
8. Resuspend in 10mL RPMI media + 10% FBS and filter with 70um filter (white)
9. Count cells: 
-If with Neubauer chamber: resuspend 1:2 (10uL cells + 10uL Trypan Blue); Calculation: average of cells per quadrant x 2 (dil) x 104 (cells/mL) x 10 (mL)
-If automatic counter: resuspend 1:10 (10uL cells + 90uL Trypan Blue); Calculation: average of two reads and take live cells x 5 (dil) x 10 (mL)
10. Resuspend pellet in right volume to: 50x106 cells/mL (10x106 cells/200uL=50,000 cells/uL) for NSG mice, or for NSG-dKO mice 100x106 cells/mL (20x106cells/200uL=100,000 cells/uL) with PBS + 2% FBS in universal tubes/epps
11. Freeze leftover cells (max 50x106cells/vial) with 10% DMSO in FBS (1mL per vial)
12. Take to the animal facility x2 the number of cells and volume needed per donor (in case there are losses due to clumping, dead volumes etc.)
13. Inject 200uL/mouse intraperitoneally
14. Follow humanization weekly by performing tail vein bleeds with heparin (applying red cell lysis buffer x3, 10min on ice) and flow cytometry for the immune markers: hCD45, mCD45, hCD19, hCD3, hCD8, hCD4, 7AAD
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Bounce PCR: optimisation free PCR for synthetic biology cloning applications.

Synthetic biology projects require the cloning of multiple DNA components, and increasingly this is done though in-vitro gene synthesis. This remains an expensive alternative to traditional cloning by PCR. However, PCR often requires time-consuming optimisation. Bounce PCR is a largely optimisation-free PCR method suitable for the rapid cloning of multiple targets. Bounce PCR is a modified version of Touchdown PCR that takes advantage of primer-extension sequences -commonly used in molecular cloning – to successfully amplify most DNA fragments without time consuming optimisation. Touchdown (and related methods) employ a sequential lowering of the annealing temperature over successive cycles to minimise non-specific primer-binding during the early amplification cycles, but maximise amplification efficiency in later cycles when the target amplicon is more abundant (Don et al. 1991; Rowther et al. 2012). However, the lower annealing temperature in later cycles may still allow for non-specific amplification. Bounce PCR takes advantage of the increased primer melting temperature caused by the addition to the template of primer-extension sequences (for example: restriction enzyme recognition sites, Gateway recombination sites, or vector overlaps for Gibson cloning) to maintain the specificity in later cycles- without sacrificing efficiency- by increasing the annealing temperature again after an initial round of touchdown.

Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., & Mattick, J. S. (1991). 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic acids research, 19(14), 4008. 

Rowther, F. B., Kardooni, H., & Warr, T. (2012). TOUCH-UP gradient amplification method. Journal of biomolecular techniques : JBT, 23(1), 1-3. 


[GUIDELINES]
This protocol describes only the thermal cycling parameters of the PCR, and should work with your preferred DNA polymerase and protocol for setting up the reaction mixture. The advantage of Bounce PCR relies on the presence of primer-extension sequences, if your primers are an exact match to your template, without 5’ extension sequences, then it is unlikely to offer an advantage over standard Touchdown PCR.

[STEPS]
SECTION: Initial denaturation
?. 
SECTION: Denaturation
?. 
SECTION: Annealing
?. 
SECTION: Extension
?. 
SECTION: Repeating
?. 
SECTION: Final extension
?.
TITLE: Colony PCR (Protocol for Thermo Scientific™ Phire™ Hot Start II DNA Polymerase)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Thermo Scientific™ Phire™ Hot Start II DNA Polymerase is a novel DNA polymerase designed for use in all routine and high throughput PCR applications. A special DNA binding domain linked to the Phire Hot Start II DNA Polymerase enhances the processivity of the polymerase, enabling short extension times and improved yields. The polymerase is also capable of amplifying long DNA fragments, such as the 7.5 kb genomic DNA used in Thermo Scientific quality control assays. Phire Hot Start II DNA Polymerase provides 2-fold fidelity compared to Taq DNA polymerase. The hot start modification of the polymerase is based on the Affibody™ inactivation method1,2 . It inhibits DNA polymerase activity at ambient temperatures, thus preventing amplification of non-specific products. At polymerization temperatures the Affibody molecule is released, rendering the polymerase fully active. Phire Hot Start II DNA Polymerase generates blunt ends in the amplification products. It does not possess the 5’→3’ exonuclease activity needed for hydrolysis experiments.</div></div>

[STEPS]
?. Add all components in a 250 μL tube making up to a 20 or 50 μl reaction.'Pro-tip': Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery. When using Phire Hot Start II PCR Master Mix, it is not necessary to perform the PCR setup on ice. 'Note': Due to the unique nature of Phire Hot Start II DNA Polymerase, optimal reaction conditions may differ from standard enzyme protocols. 'Pro-tip':Phire Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer.Add items in this order:  ABCD1Components20 μL reaction50 μL reactionFinal concentration2H2Oadd to 20 μLadd to 50 μL32X Phire Hot Start II PCR Master Mix10 μL25 μL1X4Forward primer * X μL X μL 0.5 μM5Reverse primer *X μLX μL0.5 μM6Template DNAX μLX μL7DMSO**, optional0.6 μL1.5 μL3%
ABCD1Components20 μL reaction50 μL reactionFinal concentration2H2Oadd to 20 μLadd to 50 μL32X Phire Hot Start II PCR Master Mix10 μL25 μL1X4Forward primer * X μL X μL 0.5 μM5Reverse primer *X μLX μL0.5 μM6Template DNAX μLX μL7DMSO**, optional0.6 μL1.5 μL3%
?. Gently mix the PCR reactions and transfer the tubes to a thermocycler. Thermocycling conditions for a routine PCR:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98ºC30 s13Denaturation98ºC5 s25-354Annealing*XºC5 s5Extension**72ºC10-15 s/kb6Final Extension72ºC1 min17Hold4ºC
ABCD1StepTemperatureTimeCycles2Initial Denaturation98ºC30 s13Denaturation98ºC5 s25-354Annealing*XºC5 s5Extension**72ºC10-15 s/kb6Final Extension72ºC1 min17Hold4ºC
TITLE: Collection of GBS samples at 35-37 weeks of gestation and isolation
AUTHORS: lydiariver  

[DESCRIPTION]
Pregnant women's recto-vaginal samples were collected to identify Streptococcus
agalactiae (Group B Streptococci, GBS).

[STEPS]
1. Remove each swab from the packaging and insert the swab 2 cm into the vagina, followed by the rectum, inserting the swab 1 cm through the anal sphincter. Three swabs were collected for each patient.


2. The First swab was inoculated on Todd-Hewitt broth supplemented with gentamicin 8 μg/ml and nalidixic acid 15 μg/ml (THB, Becton-Dickinson, NJ, USA). The second swab was inoculated into LIM enrichment broth (Todd-Hewitt broth with 1% yeast extract, 15 μg/ml nalidixic acid, and 10 μg/ml colistin, Becton-Dickinson, New Jersey, USA). Both swabs were incubated for 18-24 hours at 37° C in 5% CO 2 . The third swab was submerged into an appropriate transport medium (ESwab, Copan Diagnostics, Brescia, Italy). Finally, the three swabs were subcultured onto Petri dishes with Columbia agar (OXOID LTD, Basingstoke, Hampshire, England) with 5% sheep blood and incubated for 18-24 hours at 37° C to confirm GBS identification afterward.
3. Criteria for GBS identification
a) Gram-positive coccus.
b) Negative Catalase test.
c) CAMP test with wedge-shaped hemolysis in the presence of Staphylococcus
aureus ATCC 25923.
d) Positive hippurate hydrolysis.
e) Observation of growth with color change from white to orange pigment after
18-24 h incubation in the Strep B Carrot Broth® chromogenic medium (Hardy
Diagnostics®, Santa María, CA, USA).
f) Growth mauve colonies into CHROMagarTM StrepB base.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This version is based on a recipe (see<a href="https://www.protocols.io/view/human-parechovirus-a-real-time-rt-pcr-nix-assay-20-krpcv5n" target="_blank"> forked version</a>) my team and I used this assay between 2008-2015; we dubbed it the 'Nix assay'. It targets the 5'UTR and employs quite degenerate oligos.</p>
<p><em>In silico </em>sequence alignments indicated the olignucleotides could theoretically detect aft least HPeV 1-7, 17 and 18. Further validation of this recipe is ongoing.</p>
<p>However during a period of assay comparison, another assay (see link below), the 'Benschop assay' (<a href="https://www.ncbi.nlm.nih.gov/pubmed/18354819" target="_blank">J.Clin.Virol. 2008. 41(2):69-74</a>), was found to produce more sigmoidal and higher curves and 1-5 cycle improvements to C<sub>T</sub> values when compared among the sample sampe extract set.</p>

[GUIDELINES]
<ul>
<li>This protocol assumes the user is familiar with working in a laboratory, with PCR, the thermocycler and software used to run it</li>
<li>This protocol should be re-evaluated if being used with different reagents, if the oligonucleotide sequences are changed or if the cycling conditions are altered </li>
</ul>

[STEPS]
?. 
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