eamag/protocols-with-steps huggingface dataset:
TITLE: How to create baseurl for OAI-PMH harvesting with OpenRefine
AUTHORS: Andrea   Marchitelli, Carlo Bianchini, Alessandra Moi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the method for creating baseurl for harvesting an OAI-PMH repository from a list of identifiers.</div></div>

[STEPS]
?. Open the OpeRefine project containing the list of OAI identifiers prepared for harvesting.The list can be prepared by following the directions in the linked protocol How to import OAI-PMH identifiers into OpenRefine.
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?. [Creare le URL per l'harvesting]
Click on the identifier column present in the project and choose the option Edit Column > Add Column based on this column.
?. [Creare le URL per l'harvesting]
In the dialog with the new column settings enter baseurl_oai as the column name and a GREL expression that concatenates the journal's baseurl_oai, the GetRecord verb, and the OAI identifier already in the files.E.g.. 'http://www.bibliotecheoggi.it/trends/oai?verb=GetRecord&metadataPrefix=oai_dc&identifier='+valueBe careful to keep superscripts (') at the beginning and end of the URL string
?. [Test del risultato]
At this point confirming the operation you will obtain a new column called baseurl_oai containing the URLs for the GetRecord of each article.
?. [Test del risultato]
Test the result obtained by clicking on a couple of links and verifying that the corresponding page opens correctly
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Pregnant women are excluded from many, if not most, clinical trials. Although there are several potential scientific justifications for this exclusion (such as the often unknown effects of the normal physiological changes of pregnancy on drug metabolism), the primary rationale is to minimize the risk of adverse fetal effects from exposure to study-related interventions. Little formal guidance is available on how to develop pregnancy testing plans for clinical trials. To address this issue, the Clinical Trials Transformation Initiative (CTTI) conducted a project  - which included an online survey, a quantitative model, and a meeting of content experts and stakeholders - to develop recommendations for pregnancy testing in clinical research based on currently available evidence. This protocol describes the project methods. </p>

[STEPS]
?.
TITLE: CTAB genomic DNA extraction from Arabidopsis leaf material
AUTHORS: Diep R Ganguly, Pip Wilson, Gonzalo Estavillo, Xin Hou, Barry Pogson

[DESCRIPTION]
CTAB-based extraction of genomic DNA from Arabidopsis leaf tissue.

[BEFORE_START]
Ensure you grind your leaf tissue into a fine powder using mortar and pestle or Qiagen tissue lyser (place 1/8" steel ball bearing into tube with tissue sample).

Make sure leaf tissue remains frozen until the addition of CTAB buffer.

[GUIDELINES]
Gives reasonable quality and yield of gDNA. Ideal for DNA in PCRs applications, Dye-terminator Sanger sequencing reactions, and cloning. Can be used for next-generation sequencing applications, however, ensure you perform additional cleaning steps and longer centrifuge times. Run on a 1% agarose gel to ensure you have good quality and clean DNA preparation (and Nanodrop), often these preps yield a substantial portion of sheared nucleotides (likely RNA, so make sure you add RNase A during extraction).

[STEPS]
SECTION: Cell lysis
2. Aliquot required volume of CTAB buffer and heat in water bath at 60 ºC for 5-10 minutes immediately before use. 
SECTION: Cell lysis
7. Cool samples to room temperature .
SECTION: Phase separation
8. Add 300 µL chloroform and mix thoroughly with a vortex or vigorous shaking for 15 seconds. 
SECTION: Phase separation
9. Centrifuge samples for 10 minutes at 20,000 rcf.
SECTION: Precipitation
12. Add equal volume of ice-cold 2-propanol and mix by inversion.
SECTION: Precipitation
14. Centrifuge for 15 min @ 20,000 rcf.
SECTION: Resuspend DNA
16. Wash pellet with 1 mL of 70 % ethanol (mix by inversion).
SECTION: Resuspend DNA
19. Resuspend gDNA in nuclease-free H2O or low EDTA TE buffer (10 mM Tris-Cl pH 8, 0.1 mM EDTA). 
SECTION: Cell lysis
1. Prepare 2% CTAB buffer. 

 

SECTION: Cell lysis
3. Add 300 µL / 100 mg leaf tissue of CTAB buffer.
SECTION: Cell lysis
4. Add RNase A solution to a concentration 50-100 µg/mL.
SECTION: Cell lysis
5. Mix well with a vortex. Invert samples by hand to ensure that all ground tissue is in solution.
SECTION: Cell lysis
6. Incubate in water bath at 60 ºC for 30 - 60 minutes. Mix tubes periodically by inversion.
SECTION: Phase separation
10. Transfer upper aqueous (approx. 200 µL) phase to clean tube.
SECTION: Phase separation
11. Repeat steps 9-10 for a cleaner extract.
SECTION: Precipitation
13. Incubate for 30-60 min @ -20 °C
SECTION: Precipitation
15. Discard supernatant using pipette.
SECTION: Resuspend DNA
17. Centrifuge samples @ 9,200 rcf for 5 min.
SECTION: Resuspend DNA
18. Remove as much ethanol as possible using a pipette, then allow pellet to air dry for 5 minutes.
SECTION: Resuspend DNA
20. Test yield and purity of samples using a Nanodrop and running samples on a 1 % agarose gel.
TITLE: Opentrons COVID-19 testing (RT-qPCR path, Station C, 24+3+1 sample special)
AUTHORS: Max Marrone

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Prepare the reagent tube rack.In an Opentrons 24 tube rack, place the following reagents from the BP Genomics 2019-nCoV Detection Assay kit, prepared according to the kit's instructions.Well A1: Well B1: Well B3: Well D3:  (Positive Control Template diluted to 200 copies per µL, per BP Genomics' instructions)   ABCDEFGHI1Col. 1Col. 2Col. 3Col. 4Col. 5Col. 6Col. 7Col. 82Row AReaction Mix3Row BEndogenous Control MixNuclease-free water4Row C5Row DStandard curve dilution 4
[Reaction Mix]
[Endogenous Control Mix]
[nuclease-free water]
[standard curve dilution 4]
ABCDEFGHI1Col. 1Col. 2Col. 3Col. 4Col. 5Col. 6Col. 7Col. 82Row AReaction Mix3Row BEndogenous Control MixNuclease-free water4Row C5Row DStandard curve dilution 4
?. [Robot loading]
Load the labware into the robot.In Slot 4, an empty, sterile qPCR plate atop an Opentrons 96 well aluminum block atop the Opentrons Temperature Module:In Slot 5, the reagent tube rack, as prepared earlier.In Slot 3, A full, sterile Opentrons 20 µL filter tip rack:In Slot 1, the elution plate.The elution plate (a NEST 96 Well Plate 100 µL PCR Full Skirt): should have:27 samples from Station B starting at the top-left corner.Water filling the remaining wells, up to 6 columns.An extra sample in well H12 (bottom-right).  ABCDEFGHIJKLM11234567891011122A191725WW3B2101826WW4C3111927WW5D41220WWW6E51321WWW7F61422WWW8G71523WWW9H81624WWWManual samp.
ABCDEFGHIJKLM11234567891011122A191725WW3B2101826WW4C3111927WW5D41220WWW6E51321WWW7F61422WWW8G71523WWW9H81624WWWManual samp.
?. [Robot loading]
Run StationC-24-plus3-plus1-2020-04-09.py on the robot.
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?. Clean the OT-2.
?. Start pre-cooling the Temperature Module to .
6 °C
?. [Robot loading]
Wait for the run to finish.
?. [Robot loading]
Add any additional manually-prepared samples, including their master mix, to empty wells of your choosing on the qPCR plate.
TITLE: Isocitrate dehydrogenase
AUTHORS: Kimberly Chapman, Parith Wongkittichote

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isocitrate dehydrogenase is an enzyme which converts isocitrate to 2-oxoglutarate in the TCA cycle.  IDH2 uses NADP and IDH3 uses NAD.</div><div class = "text-block">Our IDH2/3 assay examines accumulation of NADH/NADPH over time. </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/e855144bdecf75d814931753c11cff3f8c6bead8b0a03fbd2d68f941d9122f3a/b65dbc5gx.png" /></div></div>

[STEPS]
?. Prepare the IDH buffer new (Place on ice)
?. To make 10 mL IDH buffer10 mM potassium phosphtate pH 7.4 (200 μL 0.5 M potasium phosphate pH 7.4), 2 mM MgCl2 (20μL 1M MgCl2),  1mM NADP (100 μL 100mM NADP)5mM isocitrate (50 μL 1M isocitrate)Add water to make 10 mL after pH ing to 7.4
?. Prepare mitochondrial lysates per protocol (will need 15-30 μg in 20 μL)
?. Load 20 μL into 96 well plate in triplicate
?. Set spectrophotometer to Kinetic, dual wavelength 340, 380 nm, 30 reads, interval 30 sec at 30°C
?. Add 200 μL of IDH buffer to each well of plate (use a multi-chamber pipetter) sitting on the holder for the spectrometer and mix by pipetting in and out 2x and start spectrophotometer
?. Calculate relative activity from the values of OD340
TITLE: Preparation of an Enriched Synaptic Vesicle Fraction
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Preparation of an Enriched Synaptic Vesicle Fraction

[STEPS]
1. Mouse brain tissue was homogenized in a buffer containing 0.32 M sucrose in PBS, supplemented with protease/phosphatase inhibitors. 






2. The homogenate was centrifuged at 1,000 × g for 10 min at 4 °C to
remove nuclei and cellular debris (P1). The supernatant (S1) was centrifuged at
15,000 × g for 15min at 4 °C to yield a crude synaptosomal pellet (P2). 
3. This pellet was hypo-osmotically lysed in water containing protease inhibitors for 5 min at 4
°C and passed through both a 22- and 27½-gauge needle (10 times each). 
4. This suspension was centrifuged at 23,000 × g for 22 min at 4 °C, and the resulting supernatant LS1(S3) was centrifuged again at 174,900 × g for 2 hours at 4 °C in a STi32 Beckman rotor. The final pellet (LP2 or P4) containing an enriched fraction of synaptic vesicles was used for subsequent
experiments.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol details the Ponisio Lab's collecting protocol for the 2017 Sky Islands season. Insects were collected into cyanide kill jars and pinned later the same day.</p>

[STEPS]
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TITLE: AlphaHOR-RES: a method for enriching centromeric DNA
AUTHORS: Nicolas Altemose, Annie Maslan, Owen Smith, Kousik Sundararajan, Rachel Brown, Aaron Straight, Aaron  Streets

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Directed Methylation and Long-read sequencing (DiMeLo-seq) can be used to map protein-DNA interactions in repetitive regions, including the centromere. If you plan to use DiMeLo-seq for centromere mapping, then centromere enrichment is a valuable technique to increase the output signal and get more data. AlphaHOR-RES is a method to enrich for centromeres using MscI and AseI restriction digest. </div><br/><div class = "text-block">Refer to "DiMeLo-seq: Directed Methylation with Long-read sequencing" and "pA-Hia5 Protein Expression and Purification" on protocols.io for the rest of the DiMeLo-seq protocol. </div></div>

[STEPS]
?. Extract genomic DNA from ~25 million cells using an NEB High Molecular Weight DNA extraction kit with 300 rpm agitation during lysis.
?. Elute DNA in 300 μL elution buffer and allow to relax at 4ºC for 2 days. DNA will remain viscous until it is digested.
?. Once fully relaxed/solubilized, add 37 μL NEBuffer 2.1, 100 units of MscI, and 100 units of AseI to reach a total volume of 370 μL in a 1.5 mL LoBind Eppendorf tube. Place the reaction on a rotator at 12 rpm and leave overnight at 37ºC.
?. Quantify DNA concentration using a Qubit Broad Range DNA kit.
?. Mix DNA with orange loading buffer and load onto a 0.3% TAE agarose gel made with Lonza SeaKem Gold agarose and 15 μL SYBRSafe gel stain per 100 mL gel. Load a GeneRuler High Range DNA Ladder in an adjacent lane. To avoid overloading, load no more than 250-300 ng of DNA per mm of lane width. Run gel at 2 V/cm for 1 hour and image over a blue light transilluminator.
?. Cut gel to remove fragments smaller than 20 kb. Keep everything larger than 20kb (up to the well itself).
?. Purify DNA from the resulting gel slice using a Zymoclean Large Fragment DNA Recovery Kit with the following modifications:
?. Melt the gel slice at room temperature on a rotator at 12 rpm.
?. Elute DNA from the column twice with elution buffer heated to 70ºC.
?. Quantify DNA concentration with a Qubit Broad Range DNA kit.
?. Prepare DNA for sequencing using an ONT LSK-109 (multiplexing) or LSK-110 (not multiplexing) native library prep kit. For a more detailed library prep, please refer to "DiMeLo-seq: Directed Methylation with Long-read sequencing" step 12.
?. Sequence on a v9.4 MinION flow cell.
TITLE: Protocols for &#34;Functional efficacy of the MAO-B inhibitor safinamide in murine substantia nigra pars compacta dopaminergic neurons in vitro: a comparative study with tranylcypromine&#34;
AUTHORS: Nicola Biagio Mercuri

[DESCRIPTION]
Methodological collection for electrophysiological investigations of neuronal firing in vitro

[STEPS]
TITLE: Stowers Institute for Medical Research Rodent Facility Environmental Summary
AUTHORS: Debra Dukes, Michael Elmore

[DESCRIPTION]
Mice (Mus Musculus) are the most common research organisms used in biomedical research. We have 
housed and maintained a variety of strains at the Stowers Institute for Medical Research since 2000.
Our environment and housing parameters provide a standardized guideline for our staff to follow.
This information assures the protection of our research using the rodent model, with proper use of 
housing, nutrition, maintenance, and environmental enrichments for the healthiest rodent specimens
for our research. The provided guideline is currently being used at the Stowers Institute in 2023.

[STEPS]
TITLE: A new protocol for multispecies bacterial infections in zebrafish and their monitoring through automated image analysis
AUTHORS: Désirée A. Schmitz, Tobias Wechsler, Hongwei Bran Li, Bjoern H. Menze, Rolf Kümmerli

[DESCRIPTION]
The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogenhost interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish. 

[STEPS]
TITLE: A Brief Look of the Evolutionary History of Cardiolipin Synthesis Pathway
AUTHORS: 181830691 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cardiolipin is an important structural and functional component of the mitochondrial intima of eukaryotic cells and the bacterial membrane of prokaryotic cells, which is considered to be one of the important evidences supporting the origin of mitochondria from bacterial endosymbiosis. However, it is generally believed that the biosynthesis of cardiolipin in bacteria and eukaryotes is catalyzed by two different types of cardiolipin synthases (CLS) with different domains, namely CLS_PLD and CLS_CAP. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Phenol/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface. Please see the <a href="http://openwetware.org/wiki/Phenol/chloroform_extraction" target="_blank">OpenWetWare</a> website for more details.<br /><br /><strong>Citation:</strong>&nbsp;OpenWetWare contributors, 'Phenol/chloroform extraction',&nbsp;<em>OpenWetWare, ,</em>&nbsp;13 September 2010, 13:37 UTC, &lt;<a class="external free" title="http://openwetware.org/index.php?title=Phenol/chloroform_extraction&amp;oldid=453981" href="http://openwetware.org/index.php?title=Phenol/chloroform_extraction&amp;oldid=453981" target="_blank">http://openwetware.org/index.php?title=Phenol/chloroform_extraction&amp;oldid=453981</a>&gt; [accessed 21 January 2015]

[STEPS]
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TITLE: Restriction enzyme & gel electrophoresis
AUTHORS: Hung Liang Pai

[STEPS]
?. [Preparation]
Set the dry bath incubator,
37 °C
?. [Preparation]
Take 10x buffer from the  to thaw.
[fridge]
?. [Protocol-Restriction enzyme]
To add  DNA for each sample into each eppendorf,  calculate the required DNA volume for each sample. The volume is decided by the formula below:Volume(sample)= / concentration measured by spectrophotometer(ng/uL)
500 ng
500 ng
?. [Protocol-Restriction enzyme]
Calculate the required ddH2O volume for each sample. The volume is decided by the formula below:Volume(ddH2O)=20uL - 2uL - 0.5uL - 0.5uL - Volume(sample) uL
?. [Protocol-Restriction enzyme]
Take sufficient eppendorfs and mark them for each sample. First, add ddH2O based on previous calculation in each eppendorf. Next, add DNA samples based on previous calculation in each eppendorf.
?. [Protocol-Restriction enzyme]
Take an eppendorf and mark as CT(cocktail). Add 10x buffer to the eppendorf. The volume is decided by the formula below: Volume(10x buffer)=2uL x (the number of samples +1)
?. [Protocol-Restriction enzyme]
Take the determined restriction enzyme from the. Add RE to the previous eppendorf respectively. Remember to put restriction enzyme  during the whole process and take RE back to theright after the process ASAP. The volume is decided by the formula below: Volume(RE 1)=0.5uL x (the number of samples +1) Volume(RE 2)=0.5uL x (the number of samples +1)
[fridge]
on ice
[fridge]
?. [Protocol-Restriction enzyme]
Addof CT in each eppendorf. Vortex and spin down each eppendorf to make the liquid stay at the bottom.
3 µl
?. [Protocol-Restriction enzyme]
Put each eppendorf in the  dry bath incubator for or even more.
37 °C
?. [Protocol-Gel electrophoresis]
Put a gel into the electrophoresis tank.
?. [Protocol-Gel electrophoresis]
Take a duran youtility. Add  TAE and  ddH2O into it and then mix it. Put it into the electrophoresis tank. Make sure the gel is soaked into the liquid completely.
3.5 mL
350 mL
?. [Protocol-Gel electrophoresis]
Take  of marker (1kb) and load it in the first well.
5 µl
?. [Protocol-Gel electrophoresis]
Take loading dye and drop on the parafilm  separately. The volume is decided by the formula below: Volume(loading dye)=1uL x (the number of samples)
?. [Protocol-Gel electrophoresis]
Takeof sample reacted with RE for . After pipetting with loading dye, add it into each well. Repeat this step until all the samples are filled into each well.
5 µl
1 µl
?. [Protocol-Gel electrophoresis]
Connect wires with the power supply. Set time for  and click "start" button. Make sure there are bubbles appearing next to anode and cathode.
?. [Protocol-Gel electrophoresis]
After gel electrophoresis, put the gel into gel reading machine and report the result.
TITLE: Cardiac Action Potential Restitution Protocol
AUTHORS: Shailesh Agarwal, Chase Fiore, Robert Harvey

[DESCRIPTION]
This protocol describes an approach that can be used to quantifiy the rate dependence of cardiac action potential duration.

[STEPS]
1. Membrane potentials were recorded from isolated pig ventricular myocytes using the whole-cell configuration of the patch clamp technique.
2. Isolated cells were placed in a perfusion chamber on the stage of an inverted microscope and bathed in an extracellular solution containing (in mM) NaCl 140, KCl 5.4, CaCl22.5, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH 7.4), maintained at 37°C.
3. Cells were patched using microelectrodes with resistances between 1 and 2 MΩ.   
4. Cells were dialyzed with an intracellular solution containing (in mM): K-aspartate 110, KCl 25, NaCl 5, MgATP 3, cAMP 0.002, phosphocreatine dipotassium 10, EGTA 0.01, and HEPES 10 (pH 7.2).
5. Membrane potential was recorded under current clamp conditions using a Multiclamp 700B voltage clamp amplifier, Digidata 1440A computer interface, and pClamp 11 data acquisition and analysis software (Molecular Devices). Data were lowpass filtered at 4 kHz, and sampled at 10 kHz.
6. Action potentials (APs) were elicited by applying a depolarizing stimulus approximately 2 ms in duration. The amplitude of the stimulus was adjusted to approximately 1.2x the minimum value needed to trigger an AP.   
7. Cells were not used if the resting membrane potential was less than -75 mV, the stimulus artifact overlapped the upstroke of the AP, or a stable baseline could not be established.   
8. AP restitution was evaluated by triggering an AP once every 800 ms for 20 beats to establish a baseline. The stimulus interval was then reduced by 20 ms each subsequent beat for 10 beats, then by 10 ms each beat for 20 beats, and then by 5 ms each beat until we observed AP duration alternans or it was no longer possible to elicit an AP.
9. Myocytes were then exposed to test drug. During this period a new baseline was established by recording APs elicited once every 5 s for approximately 5 min.
10. Drug effects were then evaluated by repeating the restitution protocol outlined in step 8.
11. Data were analyzed by measuring the duration of each AP recorded during the restitution protocol and plotting it as a function of the preceding diastolic interval (time between 90% repolarization of the previous AP and upstroke of the current AP).
12. The slope of the restitution curve was then measured as the first derivative of the polynomial fit to the data at the shortest diastolic interval.