eamag/protocols-with-steps huggingface dataset:
TITLE: APPearl Protocolo v1.0
AUTHORS: José   de Jesús Vidal-Mayo, Thierry Hernández Gilsoul, Javier Mancilla-Galindo, Ashuin Kammar-Garcia, Ramón Alejandro Loeza Güemez, Nielzer Armando Rodríguez Almendros, Omar Yaxmehen Bello-Chavolla

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Ventilación temprana en posición prono alternado en pacientes conscientes sin soporte ventilatorio invasivo con COVID-19 (APPearl)</span><span></span></div><div class = "text-block">Ensayo clínico adaptativo, multicéntrico, controlado, aleatorizado, abierto, del empleo de posición decúbito prono temprano en pacientes conscientes con COVID-19 grave sin soporte ventilatorio invasivo, para prevenir la ventilación mecánica invasiva y la muerte. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A protocol for extracting RNA from Aiptasia, modified from the Weis lab protocol by Angela Poole and Mauricio Rodriguez-Lanetty. Note that the final product has significant DNA contamination and needs to be cleaned up via DNase treatment. 
 

[BEFORE_START]
<p>Make sure to setup a clean area in the fume hood, wipe down all pipettes with RNase Away/Zap, and cool down the centrifuge.</p>

[STEPS]
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TITLE: MPAPASS Software Collection
AUTHORS: Joshua Welsh, Sean Cook, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is collection contains the protocols required for each step in the mpapass software pipeline for performing stitched multiplex analysis. This is one of a number of protocols in the pipeline for using the mpapass software package and is applicable to the latest release of the software.</div></div>

[STEPS]
TITLE: Ea(52-68) uptake assays
AUTHORS: Rebecca Wallings

[DESCRIPTION]
Ea(52-68) uptake assays on pMacs

[STEPS]
1. Reconstitute MHC-II Ea chain (Ea) (52–68) peptide (Anaspec) in sterile distilled H2O to a final concentrate of 1mg/mL. 

Once peritoneal macrophages are adhered to plates, add 5µg per well of 24-well plate and incubate  for 18-hours

Harvest cells as per flow cytometry protocol and stain with eBioY-Ae antibody (ThermoFisher) to assess Ea52-68 peptide bound to I-Ab
TITLE: Sinai SCENT TMC- Methylation Array (EPIC)
AUTHORS: Sojin Kim

[DESCRIPTION]
High throughput, proven procedure for bisulfite conversion of DNA
96-well desulphonation and recovery of bisulfite-treated DNA

[STEPS]
SECTION: High-Throughput Bisulfite Conversion of DNA
1. Add 5 μl of M-Dilution Buffer to each DNA sample in a Conversion Plate and adjust the total volume to 50 μl with water. Mix each sample by pipetting up and down.

Example: For 14 μl of a DNA sample add 5 μl M-Dilution Buffer and 31 μl water.
SECTION: High-Throughput Bisulfite Conversion of DNA
2. Incubate the Conversion Plate containing the samples at 37°C for 15 minutes.
SECTION: High-Throughput Bisulfite Conversion of DNA
3. After the above incubation, add 100 μl of the prepared CT Conversion Reagent to each sample and mix.

Note: The CT Conversion Reagent is light sensitive, so try to minimize the reaction's exposure to light whenever possible.
SECTION: High-Throughput Bisulfite Conversion of DNA
4. Incubate the Conversion Plate in the dark at 50°C for 12-16 hours (e.g., using a thermal cycler).

Note: See Appendix (page 11) for alternative incubation conditions (e.g., when using the Illumina Infinium® Methylation Assay).
SECTION: High-Throughput Bisulfite Conversion of DNA
5. Incubate the sample at 0-4°C (e.g., on ice or using a thermal cycler) for 10 minutes. Samples may be kept at 4°C for up to 20 hours.
SECTION: High-Throughput Bisulfite Conversion of DNA
6. Add 400 μl of M-Binding Buffer to each well of a Zymo-Spin™ I-96 Binding Plate on a Collection Plate.

Note: The capacity of each well of the Binding Plate is 1.1 ml. The capacity of each well of the Collection Plate is 800 μl. Empty the Collection Plate whenever necessary to prevent contamination of the Binding Plate contents by flow-through.
SECTION: High-Throughput Bisulfite Conversion of DNA
7. Load the samples (from Step 5) into the wells of the Zymo-Spin™ I-96 Binding Plate containing the M-Binding Buffer. Mix by pipetting up and down.
SECTION: High-Throughput Bisulfite Conversion of DNA
8. Centrifuge at ≥ 3,000 x g (5,000 x g max.) for 5 minutes. Discard the flow-through.
SECTION: High-Throughput Bisulfite Conversion of DNA
9. Add 400 μl of M-Wash Buffer to each well and centrifuge at ≥ 3,000 x g for 5 minutes.
SECTION: High-Throughput Bisulfite Conversion of DNA
10. Add 200 μl of M-Desulphonation Buffer to each well and let stand at room temperature (20-30°C) for 15-20 minutes. After this incubation, centrifuge at ≥ 3,000 x g for 5 minutes.
SECTION: Infinium HD Assay Methylation Protocol
11. Follow the Illumina Infinium HD Assay Methylation Protocol Guide. (see the reference)
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Reagents</strong></p>
<p>Liquid (88%) phenol</p>
<p>1% w/w Ficoll 400</p>
<p>0.5M DTT solution (77 mg/ml)</p>
<p>10M LiCl</p>
<p>10% w/v SDS</p>
<p>1% Ponceau S (dye)</p>
<p>Acetone<br /><br /><strong>Purifying proteins<br /></strong>Proteins are easily precipitated from phenol with 10 volumes of methanol. I use Ficol l as a carrier. Ficoll stabilizes the pellet and makes it visible. Four volumes of methanol efficiently precipitates all but the smallest proteins. Acetone precipitates phospholipids. Methanol won't precipitate alcohol soluble proteins (usually of little concern). Methanol precipitates proteins poorly from acidic solutions. An organic base or a buffer (e.g. ammonium acetate ) solves this problem. Adding sucrose during protein extraction conveniently 'inverts' phases.<strong> <br /><br />How efficient is phenol extraction of proteins?<br /></strong>The distribution coefficient of proteins into the phenol phase is greater than 100  (Pusztai, A. <em>Biochem J </em>99 , 93 ; 1966, in my experience greater than 1000). Hydrogen bonding to the peptide backbone is important; small peptides likely have lower <br /> distribution coefficients. Phenol is less a denaturant than a solvent. Salt (or higher pH) helps to extract histones and heat, higher pH or denaturants help to strip TMV capsid from the virion. Phenol (above, carbolic acid) was isolated from coal tar in 1834. It served as a bacteriocide in the late 19th century. Phenol extraction first served to purify (deproteinize) carbohydrates (Westphal, et al. <em>Z Naturforschung B </em>7 , 148, 1952). It was subsequently adapted to 'purify' nucleic acids (Kirby, <em>Biochem J </em>64 , 405, 1956 ). It also separates glycoproteins (Howe, et al, <em>Meth Enzymology </em>28 , 236, 1972) from erythrocyte membrane non-glycoproteins.<strong> <br /><br />Basic principles<br /></strong>Phenol dissolves proteins (for an interesting example see Cohn, E.J. and Conant, J.B. The molecular weight of proteins in phenol . <em>PNAS </em>12 , 433- 438, 1926) and lipids leaving water soluble matter (carbohydrates, nucleic acids, etc.) in the aqueous layer. Particulate and 'ambiguous' matter remain insoluble. Phenol extraction of nucleoproteins (e.g virus particles) gives pure products. Crude tissue yields complex mixtures, particularly in the aqueous phase.<strong> <br /> <br />Preparing phenol<br /></strong><br /> Phenol is often distilled and then saturated with water. Chelators (8-hydroxyquinolin e) are added to inhibit oxidation. Phenol is usually neutralized by dialyzing against a buffer. Biochemists have ignored the acidity of phenol (pK ~10) which is itself the major buffer above pH 7 (and will titrate buffers proportional to the buffer/phenol ratio). It is far simpler to neutralize phenol by adding a weak base (such as tris, I prefer N-ethylmorpholine, a liquid). Adjusting the phenol/tris ratio is more convenient (and reproducible) than dialyzing. Raising pH stimulates oxidation so neutralize phenol immediately before use. <br /><strong> <br /> <br /> <br /> <br /></strong></p>

[STEPS]
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TITLE: Consideration of the information needs of pregnant women with elective caesarean section in antenatal classes – a qualitative study from the perspective of midwives
AUTHORS: Sandra Jaax, Barbara Prediger, Nadja Könsgen

[DESCRIPTION]
 Background: Qualitative studies show that pregnant women often feel inadequately prepared for a caesarean section (CS) in antenatal classes. To date, no data have been published in Germany on the attendance of antenatal classes by women with elective CS, nor are the reasons for their attendance or non-attendance known. Antenatal classes offer pregnant women a lot of valuable information beyond the topic of vaginal birth, therefore they are also important for pregnant women with elective CS. The study will explore the demand for antenatal classes among pregnant women with elective CS from the midwives perspective, along with factors that either hinder or promote their attendance. Additionally, the study aims to assess the information needs of pregnant women with elective CS in antenatal classes from the midwives' perspective and how these information needs could be addressed better in antenatal classes as well as the extent to which (elective) CS is covered in antenatal classes.

Methods: The study population consists of midwives who practice in Germany, currently provide antenatal classes and are experienced in giving antenatal classes. In their antenatal classes, they should have experience with pregnant women with
elective CS, if possible. The study will be conducted as semi-structured expert interviews and the data will be evaluated using Mayring’s qualitative content analysis. 

Discussion: This study is an opportunity to gain a comprehensive picture from the midwives’ perspective. It will be complemented by a mixed-methods study exploring the topic from the perspective of pregnant women with elective CS. The midwives’ perspective allows for an overarching perspective including whether suggestions for improvement can be implemented. A possible selection bias in the study population may limit the results. Midwives who value CS education are more likely to participate in the study than those who do not.

Ethics: Ethical approval for the study was obtained by the Medical Ethics Committee of the University of Witten/Herdecke. Results will be published in a relevant scientific journal and communicated to interested participants (when demanded).

Ethical approval date: October 04, 2023
Estimated start of the study: October 2023

Detailed information on our protocol can be found in the attached PDF file.

[STEPS]
1.
TITLE: Carbohydrate Services
AUTHORS: BOC       Sciences

[DESCRIPTION]
BOC Sciences supports the research and development of carbohydrate synthesis with an established international reputation. BOC Sciences' advanced capabilities in carbohydrate synthesis, purification and identification have benefited our customers in academia, research institutions, biotechnology and pharmaceutical industries at all stages of R&D from basic research to clinical trials.
https://www.bocsci.com/carbohydrate-service.html

[STEPS]
TITLE: Isolated Brain Mitochondria Respiration protocol
AUTHORS: Livia Hecke Morais, Linsey Stiles

[DESCRIPTION]
Brain Mitochondria Respiration protocol. Assay developed by Dr. Linsey Stiles at the UCLA Metabolomics
Center, David Geffen School of Medicine at the University of California, Los Angeles, CA, USA

[STEPS]
SECTION: Preparation
1. The day prior to the Seahorse assay, a Seahorse cartridge needs to be hydrated overnight.

SECTION: Load the Seahorse Cartridge
4. Load 20 µL per port in the Seahorse cartridge with a multichannel pipet
SECTION: Load the Seahorse Cartridge
3. Prepare the compounds in MAS to be loaded into the Seahorse cartridge ports. Final concentrations in the well were:
a.     4 mM ADP 
b.     3 µM oligomycin
c.     4 µM FCCP
d.     2 µM rotenone and antimycin A
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
5. The prepared Seahorse cartridge needs to be calibrated prior to loading the sample plate.
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
6.      
1. Prepare template with plate map and running program. An example of an isolated mitochondria running protocol:
 
 
 
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
7.    2. Start the Seahorse Run
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
8.   3.  Load the cartridge for calibration
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
9.      
1. Prepare mitochondria sample dilutions based on the protein concentration of each sample
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
10. a.     Samples preparation includes 10X substrate, isolated mitochondria, and MAS Buffer for the desired number of wells
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
11. 2. Load 20µL of the mitochondria sample per well of the XF96 microplate
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
12.      
3. Centrifuge the plate at 2,100 x g for 5 minutes
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
13.      
4. Stop the centrifugation without a break
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
14.      
5. Add 130 of MAS per well after centrifugation
SECTION: Prepare the XFe96 Seahorse Instrument and Calibrate the Cartridge
15.      
6. Load the Seahorse plate into the instrument
SECTION: Preparation
2. All isolated brain mitochondria samples are prepared in Mitochondrial Respiration Buffer (MAS) prepared with containing:
220 mM mannitol, 70 mM sucrose, 5 mM KH2PO4, 5 mM MgCl2,
2 mM HEPES, 1 mM EGTA, and 0.1% (w/v) fatty acid-free BSA.
TITLE: CRISPR RNP Electroporation Protocol
AUTHORS: Aditya Mohan

[STEPS]
?. [Culture Cells]
Subculture cells for a minimum of 2-3 days before electroporation, and visually inspect the cells with a microscope to ensure healthy appearance.
?. [Form the RNP Complex]
For each well-undergoing electroporation, dilute the guide RNA and Cas9 enzyme in PBS gently swirling the pipet tip while pipetting:    1.3 uL of STERILE PBS    5.1 uL of GenCrispr NLS-Cas9-EGFP Nuclease     3.6 of diluted sgRNA oligos_______________________________________________________Total: 10 uL of total volume
?. [Culture Cells]
Resuspend Oligos in IDTE Buffer at 100 uM    For a 2nmol IDT order, dilute oligos with 20 uL of Tris-HCL (Dharmacon)
?. [Form the RNP Complex]
Incubate for 15 minutes at Room Temperature
?. [Prepare Nucleofector System  ]
Turn on the nucleofector and load the X-core and the program of the desired cell type (or closest cell type)
?. [Resuspend the Alt-R Cas9 Electroporation Enhancer ]
Resuspend the Alt-R Cas9 Electroporation Enhancer to 100 uM in IDTE.
?. [Prepare the Sorting Plate]
Prepare a 96 well culture plate to receive cells following Nucleofection     (Ideally U Bottom plate and at least 175 uL of culture media) and prewarm to 37 degrees C.
?. [Prepare the Quenching Plate]
Prepare a 24 well plate with at least 1 mL of media and prewarm to 37 degrees C
?. [Collect Cells]
Collect 1M cells per electroporation and MAKE SURE TO WASH THE CELLS WITH PBS.Centrifuge the cells at 30g for 10 minutes at RT
?. [Collect Cells]
Remove as much supernatant as possible without perturbing the pellet.
?. [Electroporation]
Resuspend the cell pellet in 96 uL of the Ingenio® Electroporation mediaAdd 4 ul of the diluted electroporation enhancerAdd 10 uL of the RNP gRNA mix to the well
?. [Zap Step]
Load solution into the Nucleofector module and make sure there are no air bubbles.Zap and keep in the zap mixture for 10 seconds before removing
?. [Harvest]
Remove the cells from the cuvette and  add the cells to the quenching plate for a minimum of 4-5 hours
?. [FACS Sorting]
Take samples to be sorted.
TITLE: Prima Keto UK challenge For Weight loss
AUTHORS: health  

[DESCRIPTION]
Prima Keto UK challenge For Weight loss

[STEPS]
SECTION: Prima Keto UK challenge For Weight loss
1. these medicines may help burn fat for the energy to do strenuous exercise. They may also help you lose weight. If you take these medicines daily, you may notice an increase in energy levels. They may also help reduce fatigue and weakness in your body. Taking these tablets may reduce recovery time after training.Prima Keto UK

What are the negative effects of Prima Keto UK?

Weight loss supplements claim to have no side effects. Organic and nutritious ingredients combine to form a weight loss supplement. This supplement does not have any adverse effects when taken in the right amount.

However, it is more effective to use the prescription under the direction of the manufacturer and the supervision of a doctor. It helps prevent overdose.




ViaKetoBHB The scientific basis of apple gummies

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As the most efficient way to lose weight, ketosis encourages your body to use fat as its primary source of energy. It can take weeks or months to achieve this attitude yourself. A few days after taking this pill, you will have ketosis. It takes much less time than usual to start a ketogenic diet. As a result of using fat as the main source of energy, the BHB salt in the mixture allows the body to enter ketosis. Using fat as a fuel improves energy and mental clarity and speeds weight loss.

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TITLE: Five things you need to know about PD-1 inhibitor
AUTHORS: Alex Brown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PD-1 inhibitors</span></a><span>, including PD-1 antibodies and PD-L1 antibodies, are a new drug for tumor immunotherapy. Unlike surgery, chemoradiotherapy, and targeted drugs, the PD-1 inhibitor itself does not directly kill cancer cells, but rather activates the patient's own immune system to fight cancer.</span></div><div class = "text-block"><span>The first clinical trial of PD-1 inhibitor began in 2006 and it was first officially marketed in September 2014. Since then, it has been selected by </span><span style = "font-style:italic;">Scienc</span><span>e and the American Society of Clinical Oncology as the “Biggest Progress of the Year”. The inventor of PD-1 inhibitors has also won the Lasker Award, which is known as the "Nobel Prize in Medicine". </span></div></div>

[STEPS]
TITLE: 城市微生物相調查計畫—採集土壤教學
AUTHORS: Hsin-Mao  Wu

[DESCRIPTION]
吳昕懋

[STEPS]
SECTION: 採土與iNaturalist紀錄教學
1. 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   
   

SECTION: 採土與iNaturalist紀錄教學
2. 將塑膠袋中的土均勻混合，倒入採集罐之中  
 



SECTION: 採土與iNaturalist紀錄教學
3. 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: 採土與iNaturalist紀錄教學
4. 最後拍兩張照片
 

   
        

 

SECTION: 採土與iNaturalist紀錄教學
5. 接著打開iNaturalist app
SECTION: 採土與iNaturalist紀錄教學
5.1. 如圖所示，點選右下角的加號  

SECTION: 採土與iNaturalist紀錄教學
5.2. 如下圖所示接著需選擇觀察類型，請點「選擇照片」   

SECTION: 採土與iNaturalist紀錄教學
5.3. 請點選左上角紅框處的相機圖示，上傳第二張照片；接著點選圖中央綠框處，定位採集地點  
 

SECTION: 採土與iNaturalist紀錄教學
5.4. 按下打勾處完成定位  

SECTION: 採土與iNaturalist紀錄教學
6. 上傳完成後，若是搭載Android之裝置，可在app中的詮釋資料，找到iNaturalist ID (如下圖紅框處)  



SECTION: 採土與iNaturalist紀錄教學
7. 在採集紀錄紙上填入ID，並將紀錄紙下半部，沿虛線撕下並放入採集罐中    

SECTION: 採土與iNaturalist紀錄教學
8. 記得將採集地點恢復原狀！   

SECTION: 採土與iNaturalist紀錄教學
9. 將採集罐保存於室溫，並在10日內將樣本寄至高雄醫學大學，詳細地址資訊於問卷開頭。

SECTION: 採土與iNaturalist紀錄教學
6.1. 若是搭載iOS之裝置，請在app中的觀察紀錄右上角，點選分享按鈕後，開啟瀏覽器，並在網址列末端找到iNaturalist ID
TITLE: Rab29 fast exchange mutants: characterization of a challenging Rab GTPase
AUTHORS: Rachel C. Gomez, Edmundo G. Vides, Suzanne Pfeffer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, “GTP-locked” Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel mutants that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation. </div></div>

[STEPS]
TITLE: BSCI:414 Lab2--Create  PCR Reaction
AUTHORS: Harley King

[STEPS]
?. [Attach Exisiting Primers to Plasmid in Benchling]
Select the primers icon from the right side. Select "Attach Existing". Next click "Add locations". In the popup, select "BSCI:414 Oligos" under the class root folder. Ensure a green checkmark is beside it and that it has been added to the "Use Primers in" field. Deselect "where entire primer matches" and "where primer matches exactly once on the sequence" checkmark boxes. When finished, select "Find Binding Sites". Six primers should be found. Put a checkmark next to all primers and then select "Attach Selected Primers."Click the "SEQUENCE MAP" tab and see if you can locate the positions of all six primers.
?. [Create PCR Protocol in Plasmid Description]
Select a forward and reverse primer from the six primers you added. Right click on the un-selected primers and select "Detach Primer". Make sure to keep two primers, a forward and a reverse.
?. [Create PCR Protocol in Plasmid Description]
Use the "DESCRIPTION" tab next to the "SEQUENCE MAP" tab to make a PCR schedule for your PCR reaction. Use the NEB Q5 protocol: https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491Calculate the reagents necessary for a 20ul reaction. Use can use the template here:https://benchling.com/s/seq-sbXULtIlBpW1dHuMFhIE
?. [Add Primers to Collaborative Spreadsheet]
Below the PCR schedule, @-mention the forward and reverse primers you have chosen. Calculate the expected PCR fragment length after amplification.
?. [Add Primers to Collaborative Spreadsheet]
Add the forward and reverse primer names to our collaborate spreadsheet, https://docs.google.com/spreadsheets/d/19MUe1tEnCBquPwgs5MkIgvgpck5mXkiXufY6J56CPcw/edit?usp=sharing
?. [Update Lab Notebook]
Find and open your Lab Notebook. Update your notebook to include the PCR schedule. You can copy/paste.
?. [Update Lab Notebook]
Somewhere in the lab notebook, @-mention the name of your Lab 2 plasmid. This links the work you performed today to your plasmid.
?. [Copy Plasmid to New Folder in Benchling]
In the "BSCI:414 Plasmids" under the class root folder, find plasmid "superfolderGFP/pUC57". Copy this plasmid to a new folder you create under "BSCI:414 Lab 2" using your name e.g. "F20_HarleyKing_Lab 2". Also give the plasmid a new name like "F20_HarleyKing_superfolderGFP/pUC57".