eamag/protocols-with-steps huggingface dataset:
TITLE: Canine Influenza A Subtype Identification Assay
AUTHORS: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

[DESCRIPTION]
The Canine Influenza A Subtype Identification Assay is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of canine influenza A virus (CIV) and identification of CIV H3N2, CIV H3N8 and CIV H1N1 in nasal and pharyngeal swab samples.




[GUIDELINES]
Storage and Shipping 
The Canine Influenza A Subtype Identification Assay is shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at -80°C 

Limitation:
Strict compliance with the instructions is required for optimal results.
Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
The presence of RT-PCR inhibitors may cause false negatives.
Results of Canine Influenza A Subtype Identification Assay need to be interpreted in consideration of all clinical and laboratory findings.

Quality Control:
The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA. All assays have a limit of detection (LOD95)  12 copies/l.


[STEPS]
SECTION: Reaction Setup
1. Thaw all reagents on ice.
SECTION: Reaction Setup
2. Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in the cap and keep on ice 

 

SECTION: Reaction Setup
3. Setup the Master Mix according to the following table 1:


 

SECTION: Programming the Thermocycler
4. The following fluorescence channels should be selected: ABYTM, FAMTM, JUNTM, and VICTM.
SECTION: Programming the Thermocycler
5. ROXTM should be used as a passive reference dye.
SECTION: Programming the Thermocycler
6. The standard mode should be selected. Setup cycling condition following table 2: 
  

SECTION: Results interpretation
7. Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.
SECTION: Results interpretation
8. Each assay is considered valid if the following criteria are met:
   

SECTION: Results interpretation
9. The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
TITLE: Fecal DNA extraction by bead beating
AUTHORS: Chin Yee Tan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">From Surana Lab protocols</div><div class = "text-block">This protocol is suitable for extracting DNA from either human or mouse feces. Best results will be obtained with 10-60 mg of starting material</div></div>

[STEPS]
?. For each 2ml screw cap tube,  Add Add  Add Add .
[beads]
[Phenol/chloroform]
[SDS]
[PB buffer]
?. Bead beat on Precellys, setting 2.
?. Spin down the tubes for at 4000 RPM in microcentrifuge.
?. Proceed to PCR purification kit – for the purification of up to 10 ug PCR products.
?. Label & Place a QIAquick column in a 2ml collection tube (provided).
?. Apply the aqueous top layer of your sample onto the column and centrifuge for , 17,900g (13,000 RPM), at room temperature.
?. After the spin, dump the contents of the collection tube.
?. Wash the column by adding  of Buffer PE and repeat the spin at the same conditions for.
750 µl
?. Repeat steps 7 and 8.
?. After the second wash and spin, and after dumping the contents of the collection tube, Spin the column and collection tube one last time to remove residual wash buffer for.
?. Place each QIAquick column in a clean  microcentrifuge tube.
1 ml
?. To elute DNA, add  Buffer EB (provided in kit) OR water (pH of 7.0-8.5) to the center of the Qiaquick membrane and allow the tube to sit for .
50 µl
?. Centrifuge the column for  17,900g (13,000 RPM), at room temperature.
?. Quantify with the Qubit dsDNA BR Assay kit. Alternatively, a nanodrop suffices.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This includes portions of the methods section from:</p>
<p> </p>
<p>Teytelman L, Özaydın B, Zill O, Lefrançois P, Snyder M, Rine J, et al. (2009) <a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006700" target="_blank" rel="noopener noreferrer">Impact of Chromatin Structures on DNA Processing for Genomic Analyses</a>. PLoS ONE 4(8): e6700. https://doi.org/10.1371/journal.pone.0006700</p>
<p> </p>
<p>The goal of the exercise is to note gaps and missing details. What would/should this M&amp;M section look like today?</p>

[STEPS]
?.
TITLE: Construction of mutant library
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol is used to construct mutant library of target gene with high efficiency and low false positives/negatives rate after subsequent functional screening.

[BEFORE_START]
Make sure that the template of MEGAWHOP PCR is fresh to improve the construction efficiency.

[STEPS]
SECTION: Error-prone PCR
1. Add the following reagent to a PCR tube (50μl) (Random Mutagenesis Kit by Solarbio).

 

SECTION: Error-prone PCR
2. Program the thermocycler as follows:

 

SECTION: Error-prone PCR
3. Use the palm centrifuge to mix the solution in PCR tube.
SECTION: Error-prone PCR
4. Put the PCR tube into the thermocycler and Run the program.
SECTION: Error-prone PCR
5. Using agarose gel electrophoresis to confirm if correct construct was present.
SECTION: PCR product purification
6. PCR product purified by E.Z.N.A.® Cycle Pure Kit.
SECTION: PCR product purification
7. Test the concentration and purity of DNA using NanoDrop.
SECTION: MEGAWHOP PCR
8. Add the following reagent to a PCR tube (50μl).

 

SECTION: MEGAWHOP PCR
9. Program the thermocycler as follows:

 

SECTION: MEGAWHOP PCR
10. Use the palm centrifuge to mix the solution in PCR tube.
SECTION: MEGAWHOP PCR
11. Put the PCR tube into the thermocycler and Run the program.
SECTION: MEGAWHOP PCR
12. Using agarose gel electrophoresis to confirm if correct construct was present.
SECTION: PCR product purification
13. PCR product purified by E.Z.N.A.® Cycle Pure Kit.
SECTION: PCR product purification
14. Test the concentration and purity of DNA using NanoDrop.
SECTION: DpnI digestion
15. Add the following reagents to a PCR tube (e.g. 20μl).

 

SECTION: DpnI digestion
16. Use the palm centrifuge to mix the solution in PCR tube.
SECTION: DpnI digestion
17. Incubate at 37°C for 2 hours and heat Inactivation 80°C for 20 min.
SECTION: Digestion product purification
18. Digestion product purified by E.Z.N.A.® Cycle Pure Kit.
SECTION: Digestion product purification
19. Test the concentration and purity of DNA using NanoDrop.
SECTION: Nick ligation (T4 ligase)
20. Add the following reagents to a PCR tube (e.g. 20μl)

 



SECTION: Nick ligation (T4 ligase)
21. Use the palm centrifuge to mix the solution in PCR tube.
SECTION: Nick ligation (T4 ligase)
22. Incubate the reaction at 16℃ overnight.
SECTION: Transformation
23. Transform the nick ligation product into competent cells.
TITLE: Cleavage of the Fusion Protein (TEV Protease)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">TEV Protease, also known as Tobacco Etch Virus (TEV) Protease, is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis. </div></div>

[STEPS]
?. If necessary, concentrate the fusion protein to at least .
?. Perform a pilot experiment with a small portion of your protein. For example:
?. Scale up the pilot experiment linearly for the amount of the fusion protein to be cleaved. Save at least a small sample of the uncut fusion as a negative control.
?. Check for complete cleavage by SDS-PAGE.
?. TEV Protease and the cleaved MBP contain polyhistidine tags at their N-termini. They can be removed from the cleavage reaction by immobilized metal affinity chromatography, such as Nickel or Cobalt resin, thereby isolating the target protein in the flow through.
?. Combine  and H2O to make a total reaction volume of 45 μl.
[fusion protein]
?. Add  to make a 50 μl total reaction volume.
[TEV Protease Reaction Buffer (10X)]
?. Add .
[TEV Protease]
?. In a separate tube, combine ,  and H2O to a volume of 50 μl. Do not add TEV Protease (control sample).
[fusion protein]
[TEV Protease Reaction Buffer (10X)]
?. Incubate reaction and control sample for , , and  at  (an additional reaction can be made and incubated for 24 hours at 4°C).
30 °C
?. Take  at the indicated times above and add . Take  and add  after 8 hours (or longest incubation time).
[of reaction(s)]
[SDS-PAGE Sample Buffer (3X)]
[control sample]
[SDS-PAGE Sample Buffer (3X)]
?. Incubate the SDS-PAGE samples for - at -.
70 °C
100 °C
?. Analyze them by SDS-PAGE.
TITLE: Siblings Neighbourhood Effects
AUTHORS: Lina Hedman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">This Stata code was generated for a study using a sibling design to disentangle family and neighbourhood effects on income, with contextual sibling pairs used as a control group. The sibling design helps us to separate the effects of childhood family and neighbourhood context from adult neighbourhood experiences. We use data from Swedish population registers, including the full Swedish population.</div><style>
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							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>The GeoSweden data used in this paper are third party data owned by the Institute for Housing and Urban Research, Uppsala University. Due to the sensitive nature of the data, ethical and legal restrictions apply to this data being publicly shared. These conditions are imposed by Statistics Sweden. The GeoSweden data can only be analysed in a secure safe setting, and scholars interested in accessing the data can apply to take part in GeoSweden-based collaborative research under the leadership of a researcher based at the Institute for Housing and Urban Research in Uppsala, Sweden. For further information about the data access please see</span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ibf.uu.se/research/our-researchers/geosweden/</span></a></div><style>
							  .justify:after {
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							    display:inline-block;
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							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">The Stata code was generated by Lina Hedman (Uppsala University), for a research project together with Maarten van Ham (Delft University of Technology) and David Manley (University of Bristol).</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 615159 (ERC Consolidator Grant DEPRIVEDHOODS, Socio-spatial inequality, deprived neighbourhoods, and neighbourhood effects).</div><style>
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							</style></div></div>

[STEPS]
TITLE: FTIR
AUTHORS: Alexander Röntgen

[DESCRIPTION]
Protein FTIR protocol.

[STEPS]
7. 
6.1. 
SECTION: Background scan
1.       Spot 5 µL of buffer on prism
      Acquire background scan
 
7. 
SECTION: Sample scan
2.       Spot 5 µL of protein solution on prism
      Acquire sample scan
TITLE: Citizen scientists Guides(Chinese)
AUTHORS: Hsin-Mao  Wu

[DESCRIPTION]
Soil sample, citizen scientist

[STEPS]
SECTION: 採土
5. 移除表土，取距離土表5~7公分之處的土，裝入塑膠夾鏈袋中
[示意圖片待補]
SECTION: 採土
6. 將塑膠袋中的土均勻混合，倒入HDPE塑膠罐之中
 

SECTION: 採土
8. 最後拍兩張照片
 

   
   

 

SECTION: 如何加入專案
1. 開啟Line app，將頁面切換成「聊天」，並按下頂部的掃描圖示(紅框處)
  

SECTION: 如何加入專案
2. 掃描以下QR code或點選此連結微生物公民科學
  

SECTION: 如何加入專案
3. 加入成功後會顯示以下畫面
  

SECTION: 如何加入專案
4. 接著等待採集工具包寄送即可開始採集！
SECTION: 採土
7. 在採集紀錄紙上勾選採樣地點、採樣種類，以及填寫日期、城市、鄉鎮、地點、採集者等資訊
SECTION: 採土
9. 打開iNaturalist app，上傳剛剛拍攝的兩張照片
[上傳之螢幕截圖與步驟待補]
SECTION: 採土
10. 上傳完成後，在詮釋資料中找到ID(如下圖紅框處)  
 


SECTION: 採土
11. 在採集紀錄紙上填入ID，並將紀錄紙放入HDPE罐子中
[示意圖片待補]
SECTION: 採土
12. 最後將HDPE罐子收起來並保管好
TITLE: BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction
AUTHORS: Nicholas Sherman

[DESCRIPTION]
This is a standard metabolite sample prep using chloroform/methanol and keeping the soluble (aqueous) phase. This method will produce a clean sample free of most lipids and very hydrophobic molecules. The samples are ready for MS analysis or storage dry at -80C.

[GUIDELINES]
Make sure some type of normalization is considered before starting - dry weight, cell number, etc - so equal amounts of material can be injected into the MS. This can be done by counting the same number of cells into each sample tube or weighing the same amount of tissue/stool into each tube or if larger differences you may adjust the extraction volume to account of the differences.

[STEPS]
SECTION: Liquid Samples: Urine, Plasma, Cell Media
1. To each sample containing 100 uL add 750 µL of -20°C cold Chloroform:methanol (2:1) mixture and vortex
SECTION: Liquid Samples: Urine, Plasma, Cell Media
2. Shake tubes vigorously for 30 min at 4°C in temperature temperature-controlled thermal shaker
SECTION: Liquid Samples: Urine, Plasma, Cell Media
3. Add 400 µL of water, shake vigorously, and centrifuge for 10 min at 10000 rpm for phase separation.
SECTION: Liquid Samples: Urine, Plasma, Cell Media
4. Recover the top aq. methanolic phase as metabolite mixture and transfer to Eppendorf tube.
SECTION: Liquid Samples: Urine, Plasma, Cell Media
5. Dry the soluble metabolite samples under vacuum for 3-4 h. Store at -80°C until ready for LC-MS(/MS) analysis. 
SECTION: Liquid Samples: Urine, Plasma, Cell Media
6. *Note: save the lower phase if you need to extract lipids. Go for BAF_Protocol_009. 
SECTION: Liquid Samples: Urine, Plasma, Cell Media
7. Before running, reconstitute samples in 100 µL of 0.1% Formic acid in water containing 100X diluted Metabolomics QReSS heavy labeled standards. (https://www.isotope.com/userfiles/files/assetLibrary/MET_RSCH_QReSS.pdf)
SECTION: Liquid Samples: Urine, Plasma, Cell Media
8. Prepare a QC sample with 10 µL of each sample and transfer 50 uL of each sample to autosampler vials
SECTION: Liquid Samples: Urine, Plasma, Cell Media
9. Injection volume of each sample: 10 µL
SECTION: Samples that need disruption/lysis: Tissue, Stools, Cell pellets
10. Place the sample into reinforced tubes: frozen tissue slice or lyophilized stools. For cell pellets: mix well with 50-100 uL of water transfer solution and suspended cells to reinforced tubes.
SECTION: Samples that need disruption/lysis: Tissue, Stools, Cell pellets
11. To each sample add 5 stainless steel balls, 750 µL of -20 C cold Chloroform:methanol (2:1) mixture
SECTION: Samples that need disruption/lysis: Tissue, Stools, Cell pellets
12. Disrupt cells/tissues with Fisher Bead Mill 24 (speed: 5m/s, time: 20 sec, number of cycle: 3, dwell/pause between runs: 10 sec)
SECTION: Samples that need disruption/lysis: Tissue, Stools, Cell pellets
13. Follow steps #2 to #9.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition <em>in vivo</em>. However, it has been challenging to characterize this activity, due to the lack of quantitative assays. <br /><br />Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation <em>in vitro</em>. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of ~150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins <em>in vivo</em>.</p>

[STEPS]
?. 
?. 
?. 
?. 
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?. 
?. 
?. 
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?. 
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?. 
?. 
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?.
TITLE: In-vitro GCase Activity Assay
AUTHORS: Tae-Un Han, sidranse

[DESCRIPTION]
This is version 2 of in-vitro GCase activity assay.
We optimized the assay condition to enhance sensitivity and specificity.
Both cell lysates and protein lysates prepared in M-buffer containing protease inhibitor and 0.25% TritonX can use this protocol.

[STEPS]
1. Mix 20 mL (0.852 g in 30 mL) with 14 mL (0.576 g in 30 mL) to make M-buffer (pH 5.4, no additional pH adjustment required).
2. Dissolve one protease inhibitor tablet (Roche cOmplet mini) in 10 mL, then add Triton X-100 solution to 0.25 % (v/v) (e.g. 25 µL in 10 mL) and0.2 Mass / % volume of sodium taurocholate to make active GCase buffer.
3. Set up the desired plate layout in a 384 well plate (flat bottom, black). There should be two sections, as each sample must be prepared and assayed both with and without CBE (CBE: GCase1 inhibitor).
4. Prepare 0.8 millimolar (mM) by diluting 10 millimolar (mM) in DMSO with the GCase buffer (GCase buffer:CBE = 92:8).
5. Prepare CBE-free carrier solution in the same volume of GCase buffer/DMSO (GCase buffer:DMSO = 92:8).
6. Pipette 10 µL diluted with GCase buffer into wells of a 384- well plate. Four replication sets should be run (sample concentration: 0.7 mg/mL ~ 1.2 mg/mL). Protein concentration should be adjusted to be similar between control and experiment groups using GCase buffer.
7. Add 5 µL to the CBE-positive wells or the same volume of CBE-free carrier solution to the CBE-negative wells.
8. Cover the plate with aluminum foil and briefly centrifuge. Incubate shaking:  .
9. During incubation, prepare 4-Methylumbelliferyl-B-D-glucoside (4-MU) diluent by diluting 1 M 4-Mu (338 mg per 1ml DMF) with GCase buffer to a final concentration of 2.5 millimolar (mM) (1:400 dilution).
10. After the CBE incubation, spin down the plate and add 15 µL to reach a total volume of 30 µL in each well.
11. Cover the plate with aluminum and briefly centrifuge, and Incubate shaking:  .
12. Prepare stop solution by adding 4 mL and 1.877 g up to 25 mL in water. Final concentration of glycine is 1 Molarity (M) (pH 10.5)
13. After incubation, spin down the plate again and Add 30 µL to each well.
14. Read the 4-MU fluorescence with a microplate reader (Excitation: 365 nm; Emission: 449 nm; Cutoff: 435nm; 3 reads/well).
15. GCase activity in each protein lysate can be calculated as below.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A simple protocol for RNA extraction from various types of samples (protists, fungi, bacteria, organelles, subcellular fractions, ribosomes, etc.). Compared to the commercial Trizol® procedure, it makes use of generally available chemicals without the need for columns, thus allowing concurrent extraction of transcripts of all sizes (including RNAs &lt;200 nt). Small circular DNA molecules (up to ~9 kbp) will also be efficiently isolated. The procedure is based on a previously published protocol by Rodríguez-Ezpeleta <em>et al</em>. (DOI: 10.1007/978-1-60327-136-3_3).

[GUIDELINES]
<ul style="list-style-type: disc;">
<li>Stored in a glass bottle protected from light (e.g. wrapped in aluminium foil) and kept at 4 °C, the home-made Trizol substitute solution is stable for at least 3 months. Discard if pink or purple coloration indicative of phenol oxidation appears.</li>
<li>The procedure is scalable. Its yield, as tested with spiked-in radioactively labeled transcripts of 60-900 nt, is 70-90% (in general, increasing with size). The minimal amount of RNA that could be reproducibly extracted was 10 ng (at a concentration of 200 pg/µl).</li>
<li>Note that RNA extracted using this protocol is usually not DNA-free, so DNase treatment might be necessary. Circular DNA molecules up to ~9 kbp are isolated at high yields.<a href="&quot;file:///.file/" target="_blank"></a></li>
</ul>

[STEPS]
?. 
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?.
TITLE: Yale - Total Protein
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure to determine the total protein concentration in blood, serum, and plasma. Total Protein is determined by the biuret reaction.</div></div>

[STEPS]
?. Calibrate Cobas for Total Protein analysis by running a multi-analyte calibrator and two control serum.
?. Sample handling as performed by Cobas Mira Plus     a) Pipette 5µL of sample into cuvette.     b) Add 150 µL of Total Protein Reagent.     c) Incubate at 37ºC for 10 minutes     d) Absorbance is measured at 550nm