eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<p>This protocol was designed and developed at this laboratory.</p>
<p>The assay specifically targets the 3' UTR region of DENV-2 strains and is designed as a qualitative screening test for human cases of DENV-2 infection, but not for infection due to other known DENVs.</p>
</div>
</div>

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

[STEPS]
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TITLE: DNA extraction: Campylobacter
AUTHORS: Ben Pascoe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for DNA extraction from </span><span style = "font-style:italic;">Campylobacter </span><span>isolates using the QIAGEN QIAamp DNA Mini Kit (QIAGEN, Crawley, UK). </span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is part of BioLegend's, "<a href="http://www.biolegend.com/media_assets/support_protocol/Intracellular_Staining_Protocol_082615.pdf" target="_blank">Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets</a>".</p>

[GUIDELINES]
<p><strong>Application Notes </strong></p>
<p> </p>
<p>1. Activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced). For cytokine and chemokine detection, it is critical to include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701) in the last 4-6 hours of cell culture activation. The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining.</p>
<p> </p>
<p>2. Different cytokines/chemokines have different production peaks. In order to obtain optimal staining signals, the stimulation conditions for each stimulant need to be optimized.</p>
<p> </p>
<p>3. Some antibodies recognizing native cell surface markers may not bind to fixed/denatured antigens. For this reason, it is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/permeabilization and staining of intracellular targets. Altering the procedure such that cells are fixed prior to staining of cell surface antigens requires that paraformaldehyde-denatured antigen reactive antibody clones be empirically identified.</p>
<p> </p>
<p><strong>Note</strong></p>
<p> </p>
<p>To confirm specific anti-cytokine staining, a blocking experiment is recommended in which cells are fixed/permeabilized then preincubated with an excess amount of unlabeled anti-cytokine antibody and/or the recombinant cytokine of interest is preincubated with fluorophore-conjugated anti-cytokine antibody before its addition to the cells.</p>
<p> </p>
<p><strong>Related Information</strong></p>
<p><br />1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097.<br />2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294.<br />3. Jung T, et al. 1993. J. Immunol. Methods 159:197.<br />4. Prussin C., et al. 1995. J. Immunol. Methods 188:117.<br />5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219.</p>
<p> </p>
<p><strong>Reagent List</strong><br />1. Cell Staining Buffer (BioLegend Cat. No. 420201)<br />2. Monensin (BioLegend Cat. No. 420701)<br />3. RBC Lysis Buffer (BioLegend Cat. No. 420301)<br />4. Brefeldin A (BioLegend Cat. No. 420601)<br />5. Fixation Buffer (BioLegend Cat. No. 420801)<br />6. Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002)<br />7. Cyto-Last™ Buffer (BioLegend Cat. No. 422501)</p>

[STEPS]
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TITLE: Yale - Aspartate Amino Transferase
AUTHORS: Gary Cline, John Stack

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to measure the activity of Aspartate Amino Transferase (AST). AST activity is measured by the enzymatically coupled reactions of AST (to form oxaloacetate from aspartate and α-ketoglutarate) and malate dehydrogenase (conversion of oxaloacetate to malate with oxdiation of NADH to NAD). The rate of NAD formation is monitored by the change in absorbance at 340 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Aspartate Amine Transferase Activity analysis by running twoassayed control serum.
?. Sample handling as performed by the Cobas Mira Plus.    a) Pipette 16 µL of sample into a cuvette slot.    b) Add 145 µL of Aspartate Amino Transferase Reagent.    c) Mixture is incubated at 37˚C and spun for 10 minutes.    d) Absorbance is measured at 340 nm.
TITLE: Colony PCR for 2022 iGEM
AUTHORS: Team Fudan iGEM

[DESCRIPTION]
Based on manual included in Vazyme Taq product

[STEPS]
1. Select colonies on the agar plate to pick. Culture portion of the colony in LB media with antibiotic and grow in 37 degree. Put the remainder of the colony into a correspondingly numbered PCR tube with 20 µL of reaction mix (as below), and pipetting to complete resuspend the bacteria.
2.  
ComponentVolume/ulFinal Concentration2XT5 Super PCR Mix5ul1x10uM Forward Primer0.4ul0.4uM10uM Reverse Primer0.4ul0.4uMTemplate DNA1ul<1ug   ddH2OAdd to 10ul　Total10ul　
Primers used for screening crt genes insertion are:
 

3.  
Temperaturetime98℃03:0098℃00:10Tm-5℃00:1072℃20s/kb72℃02:004℃∞
 
4. Agarose gel electrophoresis of PCR products. One lane on the gel is loaded with 6 uL D2000 DNA ladder. Run the gel at 100 V for 20-30 minutes, and examine the gel under UV.


5. The correct bacterial clones are sent for Sanger sequencing. Once verified, these clones would be used for further experiments.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Separates molecules based on size.

[BEFORE_START]
Have a DNA Sample ready, typically either from PCR or a recently performed Restriction Digest. Dilute down the 50X TAE Buffer to 1X.

[STEPS]
SECTION: Prep Work
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SECTION: Prep Work
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SECTION: Loading the Gel
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SECTION: Loading the Gel
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SECTION: Running the Gel
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SECTION: Staining
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SECTION: Destaining
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SECTION: UV exposure
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The RT and amplification steps for SMARTseq2, as modified for previously extracted RNA slices.

[BEFORE_START]
Thaw RNA samples on ice, as well as:<br /><br />* First Strand Buffer (-20&ordm;)<br />* DTT (-20&ordm;)<br />* dNTPs (-20&ordm;)<br />* oligo dT30VN primer (-20&ordm;)<br />* Betaine (4&ordm;)<br />* TSO (-80&ordm;)

[STEPS]
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TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: U Mass - Amylase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Amylase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Canine/feline serum or plasma deproteinization for  amino acid analysis on Biochrom
AUTHORS: Amanda  Blake, Jan Suchodolski

[DESCRIPTION]
This protocol describes the deproteinization process that all canine and feline serum and plasma research samples undergo prior to amino acid analysis with a Biochrom 30+ Amino Acid Analyzer at the Gastrointestinal Lab, Texas A&M University. L-norleucine is used as an internal standard.

[STEPS]
1. Aliquot 250 μl serum or plasma into 1.5 ml microcentrifuge tube.
2. Add 250 μl [5% Sulfosalicylic acid (w/v), 500 μM L-Norleucine] to 1.5 ml microcentrifuge tube. Note: if less than 250 μl serum or plasma is available, can use as little as 100 μl and add reagent in 1:1 (v/v) ratio. i.e., if using 150 μl serum, would add 150 μl [5% Sulfosalicylic acid (w/v), 500 μM L-Norleucine].
 

 

3. Vortex each on high speed for 5-10 seconds, and then store in 4°C for 10 minutes.
4. Centrifuge 1.5 ml tubes at 10,000 rcf, 4°C for 5 minutes.10000 x g 
 


5. Transfer supernatant to centrifuge filter tubes, being careful not to disturb pellet or floating lipid layer.


 



6. Centrifuge filter tubes at 10,000 rcf, 4°C for 5 minutes. 10000 x g  Discard filter and store filtrate at —80°C until analysis.
 



2.1. To make SSA solution, weigh 5 g sulfosalicylic acid into graduated cylinder, add 6.6 +/- 0.1 mg L-Norleucine, then fill to 100 mL with Biochrom lithium loading buffer. Stir briefly on magnetic stir plate until all solids are fully dissolved. Store at 4°C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathers via LC-MS/MS was implemented based on techniques previously published by other authors: </p>
<p><span lang="EN-US">1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017.</span></p>
<p><span lang="EN-US">2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21.</span></p>
<p><span lang="EN-US">3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81.</span></p>
<p><span lang="EN-US">4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11.</span></p>
<p><span lang="EN-US">5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78.</span></p>
<p><span lang="EN-US">6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54.</span></p>
<p><span lang="EN-US">7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62.</span></p>
<p> </p>
<div>
<div>
<p><span lang="EN-US">The method is based on a solid-liquid extraction with organic solvents. The analyte was concentrated using a water bath at 40-50°C under a mild nitrogen flow. </span><span lang="EN-US">For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 software package (SCIEX, Framingham, Massachusetts).</span></p>
</div>
</div>

[BEFORE_START]
<p>Liver samples must be ground in an food processor before proceeding to extract the analytes to ensure their homogeneity of the samples. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Lawrence, J. E., and G. F. Steward. 2010. Purification of viruses by centrifugation, p. 166–181. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_166.pdf" target="_blank">published manuscript</a> for additional information.

[STEPS]
TITLE: Urea-mediated dissociation alleviate the false-positive Treponema pallidum-specific antibodies detected by ELISA
AUTHORS: qiang wang, Yan Lei, Xiaolan Lu, Guangrong Wang, Qin Du, Xiaolan Guo, Quming Fan, Guoyuan Zhang, Dongsheng Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The serological detection of antibodies to </span><span style = "font-style:italic;">Treponema pallidum</span><span> is essential to the diagnosis of syphilis. However, for the presence of cross-reaction, the specific antibody tests [e.g., enzyme-linked immunosorbent assay (ELISA)] always have false-positive results. In this study, we derived and validated the dissociation of urea in an attempt to alleviate the situation of false-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span> detected by ELISA. Six serum samples that were false-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span>detected by ELISA, and 16 control serum samples (8 sera positive for both specific IgG and IgM, and 8 IgG-positive and IgM-negative sera) were collected to select the appropriate dissociated concentration and time of urea. Our goal was to establish improved an ELISA method based on the original detection system of ELISA. The sensitivity of the improved ELISA was evaluated by 275 serum samples with class IgM-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span>. At 6 mol/L with 10 minutes dissociation of urea, 6 samples with false-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span> were converted to negative, and compared with true-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span>. The sensitivity of the improved ELISA was 100% by detecting the class IgM-positive antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span> in sera of patients with syphilis. Considering the importance at the diagnosis of syphilis, antibodies to </span><span style = "font-style:italic;">T. pallidum</span><span>in serum samples should be retested by the improved ELISA method to avoid false-positive results. </span></div></div>

[STEPS]
?. Design the experimental steps.
?. Collect clinical samples to meet the needs of the experiment, especially  false positive samples detected by ELISA.
?. Using different methods to detect TP antibodies, and to understand the characteristics of different samples, so as to facilitate grouping.
?. Pre-experiment to explore the basic conditions of urea dissociation.
?. Formal experiment to select the best  condition of urea dissociation.
?. Sensitivity evaluation of the improved TP-ELISA.
TITLE: U Mass - Surgery – jugular vein cannulation
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Chronic indwelling catheter is placed in the jugular vein for intravenous infusion and injection of drug, hormones, and metabolites. Surival surgery is performed in anesthetized mice, and mice recover from surgery after 4~5 days.</div></div>

[STEPS]
?. Anesthetize mice with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight).
?. Check the level of general anesthesia using a pinch stimulus of animal's tail and/or foot.
?. Make a transverse incision (~0.5 cm) over the trachea, and isolate the right jugular vein.
?. Carefully introduce a silastic catheter (PE 10) into the vessel. The catheter should be filled with a saline solution containing heparin (10 U/ml) and plugged.
?. Guide the catheter to the back of the neck, and place under the back skin to prevent its accessibility from the mouse.
?. Tie a silk to the catheter, and make a small opening at the back of neck.
?. This silk, which is partially exposed, will be used/pulled on the day of experiment to expose the catheter.
?. On the day of experiment, flush the catheter using a heparinised saline solution.
?. All surgical procedures should be performed using standardized aseptic techniques, and all surgical tools should be autoclaved following the surgery.
?. Apply post-operative care based on the institution’s animal care and use committee standard operating policy.
TITLE: SEWRL RS &amp; GIS Team GPS Data Collection Storage Procedures
AUTHORS: marshall.bennett

[DESCRIPTION]
Process for downloading and  managing GPS data collection projects to centralize GPS Data for analysis

[STEPS]
1. Collect data in the field. 
2. Download GPS Data to T:\sandbox\GPS_Data_Dump in an appropriate folder location for each project or data collection. 
3. Fill out record in "Read Me" Document
TITLE: Image J Nerve Density Percentage Analysis
AUTHORS: Elizabeth Smith, Jacqui Potter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ImageJ Software can be used to find the percentage of nerve density in regions of interest. These regions of interest are imaged, and the nerve percentage is found by adjusting the image's histogram threshold.</div></div>

[STEPS]
?. Open Image
?. Dowload ImageJ Software
?. File>Open>Locate desired image file>Click desired individual image (JPEG or TIFF)
?. Prepare Image for Analysis
?. Image>Type>8-bit
?. Remove over exposed areas, scale bar etc. by clicking on desired shape in Shape Tool Bar under File Tool Bar.
?. Analyze Image
?. Image>Adjust>Threshold
?. Move both slider bars to the far right. Slowly move the top slider bar to the left until you just fill in the areas of interest with "red". The percentage that appears under the histogram is the percentage of area filled. That is the nerve density percentage.
?. Reset the threshold slider bars as many times as desired. Typically, three data points which are then averaged.
TITLE: STC-1 Passaging and PD Associated Microbe Stimulation Protocol
AUTHORS: Malu G Tansey

[DESCRIPTION]
STC-1 Passaging and PD Associated Microbe Stimulation Protocol

[STEPS]
SECTION: Passaging &amp; Plating Protocol
1. Cells are passaged when they reach approximately 70% confluence

Warm trypsin, 1XPBS, and complete medium in water bath for 15 min.
SECTION: Passaging &amp; Plating Protocol
2. Aspirate off culture medium
SECTION: Passaging &amp; Plating Protocol
3. Rinse cells with 10mL D-PBS
SECTION: Passaging &amp; Plating Protocol
4. Add 3mL Trypsin to flask. Place flask back into 37°C/5% CO2 incubator for 7 minutes.
SECTION: Passaging &amp; Plating Protocol
5. Observe cells under microscope to ensure they have detached from the bottom of the flask. 

a. These cells are particularly sticky, so giving them a nice tap may be necessary to detach them.
SECTION: Passaging &amp; Plating Protocol
6. Add 9mL of complete medium to flask and transfer cell suspension to a 15mL tube.
SECTION: Passaging &amp; Plating Protocol
7. Spin down at 130g for 5 min
SECTION: Passaging &amp; Plating Protocol
8. Aspirate off supernatant and resuspend cell pellet in 1mL culture medium.
SECTION: Passaging &amp; Plating Protocol
9. Count cells
SECTION: Passaging &amp; Plating Protocol
10. Prepare cell suspension at a concentration of 3x105 cells/mL
SECTION: Passaging &amp; Plating Protocol
11. Add 1mL cell suspension per well in 12 well plate(s)
SECTION: Passaging &amp; Plating Protocol
12. For PD associated microbe stimulation, allow cells to rest for approximately 24 hours,
SECTION: Passaging &amp; Plating Protocol
13. Treat STC-1s with 10 colony forming units (CFU) of PD associated microbe per STC-1 cell
SECTION: Passaging &amp; Plating Protocol
14. Incubate for desired time and then collect conditioned media and/or cells for downstream analyses  
SECTION: LPS Pre-Treatment
15. Following plating, allow cells to rest for 24 hours
SECTION: LPS Pre-Treatment
16. Treat cells with 10ng/mL LPS for 24 hours
SECTION: LPS Pre-Treatment
17. 24 hr after LPS treatment, stimulate cells with 10 CFU of PD associated microbes per STC-1 cell
SECTION: LPS Pre-Treatment
18. Incubate for desired time and then collect conditioned media and/or cells for downstream analyses
TITLE: XPRIZE SANATA Protocol for Swab LFIA Test
AUTHORS: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Heidi Abdilla, Reda Fayek, Michelle Feng, Stephanie Lim, Amanda Naaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure outlines the protocol using any swab suitable for a SARS-CoV-2 sample collection. The purpose of this test is to detect low levels of SARS-CoV-2 antigen at a higher sensitivity. Precision Biomonitoring Inc. developed an ultra-rapid digital, disposable, highly-sensitive and inexpensive testing device used for screening purposes. The mobile app complementary to this medical device is connected through Bluetooth. Using this innovation, the patients can be tested at the point-of-care (POC), and obtain qualitative results. It is recommended that positive results should be confirmed by RT-PCR.</div></div>

[STEPS]
?. [Collecting and Preparing Sample]
Collect sample using any swab suitable for SARS-CoV-2 sample collection. For example:
?. [Collecting and Preparing Sample]
Rinse the used swab inby submerging the swab in the lysis buffer and rotating 5-7 times against the tube walls. Discard the swab in a biohazardous waste container.
?. [Using Testing Device]
Using apply   (3 drops) of the contents of the SWAB Lysis Tube into the sample port of the
80 µl
?. [Using Testing Device]
Place device on a flat surface. Let sample mixture run undisturbed for  at  .
0 Room temperature
?. [Using Testing Device]
Ensure the smartphone is connected to the testing device through Bluetooth and read the results using the mobile phone app.
The result will appear on screen as positive, negative, or inconclusive. If the result is inconclusive, conduct another test with a new device.
?. [Setting up the test]
Ensure the smartphone is fully charged and Bluetooth on the smartphone is turned on. The mobile app should be downloaded and ready to run.
When working with human bodily fluids, pathogens may be present. Wear the correct personal protection equipment (ie.gloves) and wash hands immediately after removing the gloves.