eamag/protocols-with-steps huggingface dataset:
TITLE: U Michigan - Retinal Vascular Permeability
AUTHORS: David A. Antonetti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">The vascular permeability is quantified by measuring albumin leakage form blood vessels into the retina. Fluorescent dye (FITC-BSA) is used to measure the breakdown in blood-retinal barrier to be detected when increased vessel leakage is extravasated into the interstitial space.</div></div>

[STEPS]
?. Weigh animal and record body weight for anesthetic and dye injections
?. Anesthetize animal with ketamine/xylazine mixture
?. Make an incision on skin inside of the hind leg and carefully tear away the membranes to isolate the femoral vein
?. Inject FITC-BSA into the femoral vein at 2 µl/g body weight (equal to 200 mg/kg body weight) using a 31-gauge 0.3 c.c. insulin syringe (vortex FITC-BSA before use)
?. Apply pressure on the injection site with a sterile gauze or cotton swab to stop bleeding
?. Staple the incision site and allow FITC-BSA to circulate for 2 hours
?. Anesthetize the animal again with ketamine/xylazine
?. Open the abdomen and draw 0.3 ml blood from the vena cava with a 27-gauge 1 c.c. syringe
?. Remove needle and expel blood into a heparinized micro-cuvette
?. Mix blood sample by gently reversing the tube several times and keep on ice
?. Open the chest cavity, cannulate the heart with a blunt end 22-gauge needle into the left ventricle and incise right atrium to release pressure
?. Perfuse with saline (warmed to 37°C) at 20 ml/min via the left ventricle for 2 minutes
?. Harvest the retina and place in a pre-weighed microcentrifuge tube. Rinse the harvest tools between samples to avoid cross contamination
?. Centrifuge the blood sample at 2,000 x g at 20°C for 15 minutes to separate plasma
?. Transfer the plasma to a new microcentrifuge tube and store at -80°C
?. Dry the retina samples with a Speed-Vac overnight
?. Weigh the microcentrifuge tube containing the dry retina to obtain the dry retina weight
?. Add 100 µl of 1% Triton-PBS to each retina and shake overnight to extract FITC-BSA
?. Vortex briefly, centrifuge the microcentrifuge tubes at 17,000 x g for 30 minutes and transfer supernatant to a new microcentrifuge tube
?. Dilute the plasma samples with 1% Triton-PBS
?. Dilute the stock FITC-BSA (100 mg/ml) with 1% Triton-PBS and make serial dilutions to obtain the FITC-BSA standards
?. Measure the standards (0.156 to 10 µg/ml), retina extract and diluted (1:900) plasma samples (20 µl/well, triplicate) in a 384-well black/clear bottom plate with a fluorescent plate reader (excitation 488 nm, emission 520 nm)
?. Calculate the FITC-BSA concentration of each sample with the standard curveNOTE: The auto-fluorescence background of the retina from animal without FITC-BSA injection should be subtracted for the permeability calculation.Additional tip that improves efficiency.
TITLE: Risk of pregnancy complications in living kidney donors: a meta-analysis
AUTHORS: Ioannis Bellos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Living kidney donation is growing, especially among young women. However, the risk of subsequent pregnancy complications remain unclear. The present meta-analysis aims to clarify whether kidney donation is linked to higher rates of preeclampsia, gestational hypertension, gestational diabetes, preterm birth, low birthweight or fetal death. Current literature will be gathered and pooled, while the quality of evidence will be appraised.</div></div>

[STEPS]
?. Objective
 To determine whether living kidney donation is associated with higher risk of developing maternal or fetal complications in subsequent pregnancies.Eligibility criteria
 The rate of pregnancy complications will be compared among women with history of living kidney donation and non-donors. The outcomes of interest will be: preeclampsia, gestational hypertension, gestational diabetes, cesarean delivery rate, preterm birth (Literature search
 Literature search will be performed by systematically searching from inception PubMed, Scopus, Web of Science and CENTRAL (Cochrane Central Register of Controlled Trials). In addition, Google Scholar will be screened to provide grey literature coverage, while the full reference lists of the included studies will be examined to recognize potential missing articles. No date/language restrictions will be applied.Data extraction
 The following data will be extracted: year of publication, country, eligibility criteria, sample size, study design, data source, years of donation, participants’ age at pregnancy and at donation, as well as the necessary data for the outcomes of interest.Quality assessment
 The risk of bias of the included studies will be evaluated with the ROBINS-E tool, which takes into account the following domains: confounding, selection of participants, classification of exposures, departures from intended exposures, missing data, measurement of outcomes and selection of the reported results.Data analysis
 Confidence intervals will be set at 95%. Random-effects models will be fitted using the Paule-Mandel method for between-study variance estimation. Pool estimates of odds ratios will be calculated. The inter-study heterogeneity will be quantified by the inconsistency index (I2), while the 95% predictive intervals will be calculated to assess the effects to be expected by future studies. Subgroup analysis is planned based on whether appropriate participant matching was applied or not. Funnel plots will be constructed and the Egger’s test will be performed to assess their asymmetry, if appropriate (>10 studies).Quality of evidence
 The quality of the existing evidence will be appraised following the GRADE approach. Specifically, evidence will be classified as very low, low, moderate or high by judging the following domains: study limitations, consistency, directness, imprecision and publication bias.
TITLE: AlphaFold 3 screen
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol is about the AlphaFold 3 screen.

[STEPS]
1. Protein sequences were downloaded from the Uniprot server.  
2. We accessed AlphaFold 3 from its virtual server (https://alphafoldserver.com) to run pairwise predictions with 5 models per prediction.
3. Predictions with an ipTM score of > 0.5 were considered putative hits and diagnostic plots (PAE plot and pLDDT plot) as well as the generated structures were manually inspected.
4. Predicted structures were visualized with ChimeraX-1.8.
TITLE: Blunting Protocol for NEB PCR Cloning Kit (E1202)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the blunting protocol for NEB PCR Cloning Kit (E1202).

[BEFORE_START]
Reaction volume may be scaled up or down as necessary.

[GUIDELINES]
Reaction volume may be scaled up or down as necessary. 

PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis. 

Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1.1, 2.1, 3.1, and CutSmart™ Buffer in addition to NEBuffers 1-4, BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 and 1.1 where the total yield is about 50% of optimum.

[STEPS]
1. Mix the following components in a sterile microfuge tube:  
 

2. Determine whether your reactions are using DNA digested by restriction enzymes or are sheared/nebulized or PCR products and move forward with the following steps:
 ether you are using 
 
4. Immediately inactivate enzyme in the blunting reaction by heating at 70 °C for 10 min. 
5. Proceed directly to the ligation step using the Quick Ligation Kit (NEB #M2200) or standard T4 DNA Ligase (NEB #M0202).   
 
3. Incubate the reactions with sheared/nebulized DNA or PCR products at 70 Room temperature for 30 min.   
3. Incubate the reactions containing restriction enzyme digested DNA at 70 Room temperature for 15 min. 
4. Immediately inactivate enzyme in the blunting reaction by heating at 70 °C for 10 min. 
5. Proceed directly to the ligation step using the Quick Ligation Kit (NEB #M2200) or standard T4 DNA Ligase (NEB #M0202).
TITLE: Live-cell imaging for synaptic vesicle precursors in human iNeuron axons
AUTHORS: Dan Dou, Erika Holzbaur

[DESCRIPTION]
Here, we describe procedure and equipment used for live-imaging of synaptic vesicle precursors. This was performed using DIV21 human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on scheduled upgrades to microscopy equipment during the course of this study.

[STEPS]
1. 
 

Image human iNeurons on DIV21, 48-72 hours after transfection with PGK-mScarlet-synaptophysin.
2. Replace culture media with low fluorescence imaging media.
2.1. For iNeurons, use Hibernate A medium supplemented with:

 

3. Image using spinning disk confocal microscope under 60x magnification (oil immersion objective). See “Materials and Methods” for specific microscopes and cameras used.
4. Identify axons of transfected neurons based on morphological parameters. (Boecker et al., 2020; Kaech and Banker, 2006). For example, axons can most reliably be identified by their length and should span over at least 500 µm. 
5. Identify the neuronal soma and measure ~100-150 µm from the soma. Create an ROI that includes a segment of the axon that is in the same Z plane. Image several frames at this field of view prior to photobleaching. Perform one photobleaching cycle with the 405 nm laser for 3 ms/pixel. Dramatically decreased mScarlet-SYP signal should be observed in the ROI following photobleaching, and entry of mScarlet-SYP+ vesicles into the bleached region should be readily observed.
6. Acquire time lapse recordings at a frame rate of 5 frames per second for 5 min .
TITLE: Rotarod Test
AUTHORS: Jhodi Webster

[DESCRIPTION]
The purpose of this protocol is to assess mice for coordinated movement. Mice who have degeneration within the basal ganglia pathway will display slower movement and fall off the moving rod sooner.

[GUIDELINES]
For analysis:

      The numbers you will put into prism is the time it takes for the mouse to fall off the rotarod (latency to fall). This should be recorded in seconds. 
      Since mice are measured for 5 days, you will graph data as a repeated measures to see the latency to fall over the course of 5 days. Your end graph should look like the one below:
      
 


 Your statistical test will depend on the number of groups you have. Please consult with a biostat person before publishing results.  


[STEPS]
SECTION: PROCEDURE
1. Turn on rotarod and start up rotarod software.
SECTION: PROCEDURE
2. Rename the file. I would recommend having a different file name every day you test. This will make analysis easier later.
SECTION: PROCEDURE
3. Rename the subject names and reset the lanes (on the actual rotarod).
SECTION: PROCEDURE
4. Set the speed to 4.0 to 40 RPM.
 - Over the course of 300 seconds the rotarod will accelerate from 4 to 40 RPM.
SECTION: PROCEDURE
5. Click the “Reset” button on software and the rotarod will begin turning. 

Let the mice sit for 1 hour listening to the rotarod. They need to get used to the sound of the machine.
SECTION: PROCEDURE
6. After the 1 hour, place the mice on the appropriate lane on the rod. Once all the mice are on the rod, click “On/Accel” button and the instrument will begin to accelerate.
SECTION: PROCEDURE
7. The rod will begin to rotate. As the mice fall off the lane timer will stop. If a mouse hangs on the rod and rotates with the rod for one rotation stop that lane’s timer. If a mouse is still running on the rod after 180sec, stop the timer and record the time as “180”.

I have noticed with my mice, that at 180 they are basically running, if the mouse has motor defects they will have fallen off by then.
SECTION: PROCEDURE
8. When all the timers have been stopped, the software will automatically save on the computer in a txt file. Make sure to have a flash drive to save data on.
SECTION: PROCEDURE
9. Place mice back in their home cage and start with the next cage.
SECTION: PROCEDURE
10. Do three trials with 30 min break between trials.
SECTION: PROCEDURE
11. Repeat all steps for 4 to 5 days. The mice will learn to stay on the rod over time, but if the mouse is suffering from motor defects they will struggle to stay on the rod.
SECTION: PROCEDURE
12. From the txt file acceleration and latency to fall time will be recorded.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>February, 2012, based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F, phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)</p>

[GUIDELINES]
<p>To avoid possible RNA degradation, try to work quickly in all steps.</p>

[STEPS]
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TITLE: Transfection of Naegleria gruberi
AUTHORS: Anastasios D.. Tsaousis, Eleanna Kazana

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

[STEPS]
?. [Harvest Naegleria cells that have been "transformed" to flagellates, via the protocol (see below).]
Use a 100% confluent 25cm2 flask for each transformation. https://www.protocols.io/view/flagellar-transformation-of-naegleria-gruberi-hnhb5b6
?. [Pellet the cells by centrifugation at 1000 x g for 5 minutes]
?. [Wast the pellet with ice-cold sterile 1X PBS]
?. [Pellet the cells by centrifugation at 1000 x g for 5 minutes]
?. [Wash the pellet with SM ice cold buffer. Filter sterilise an aliquot before use.]
1X SM BUFFER: 500 ml5 Mm KCl: 0.18 g15 mM MgCl2 : 710 mg120 mM Na2HPO4/NaH2PO4, pH 7.2 : 60 ml25 mM succynil acid: 1.475 g25 mM mannitol: 2.275 gFilter aliquots before electroporation
?. [Pellet the cells by centrifugation at 1000 x g for 5 minutes]
?. [Resuspend the pellet in 400 μl of SM ice-cold buffer per electroporation sample]
?. [In an 1.5 ml eppendorf tube add 10 μl of plasmids (pNaegNEO and pNaegHYG plasmids; concentration 400 ng/μl) and the 400 μl of cells in SM ice-cold buffer (above)]
?. [Incubate on ice fo 5 minutes]
?. [Transfer the suspension in an ice-cold bio-rad electroporation cuvette; 0.4 cm.]
?. [Electroporation shock: clean the ice and water of the cuvette with tissue and put them in the chamber of the BioRad Gene Pulser xCell (see attached]
?. [Electroporation conditions for the BioRad Gene Pulser xCell: 175 V, 500 μF, 400 Ω.]
?. [Remove the cuvette from the chamber and put back on ice and incubate fo 5 minutes]
?. [In a 24 well plate, add 400 μl of the electroporation (above) per well and 1,400 μl of M7 media supplemented with 10% FBS and 1% penicillin/streptomycin]
M7 rich media for growing Naegleria gruberi axenically contains 0.54 % glucose (Fisher Scientific  – Product No.  G/0500/61), 0.5 % yeast extract (Melford), 0.0045% L-methionine (Duchefa Biochemie – Product No. M0715.0100) and 2% KPi buffer solution containing 0.18% KH2PO4 (Melford  – Product No. P0574) and 0.25 Na2HPO4 (Melford  – Product No. S2002) , 10% FBS (Sigma  – Product No. 7524 non-USA origin, sterile-filtered, suitable for cell culture) and 1% penicillin/streptomycin (Gibco from Fisher Scientific  – Product No. 11548876).
?. [Seal the 24 well plate with parafilm and incubate at 32oC for 24-48 hours.]
?. [After maximum of 48 hours add the corresponding selectivedrug to the transected cells (300 μg/ml of Hygromycin B or 700 μg/ml of Neomycin (G-418))]
?. [Incubate at 32oC for 24 hour]
?. [Carefully remove the supernatant (along with dead cells) and add 2 ml of fresh media with the corresponding selective drug (Hygromycin B or G418) to the final concentrations shown above.]
?. [Incubate at 32oC for 72 hours]
?. [Transfer the cells in a 25cm2 flask and incubate at 32oC and analyse them by fluorescence microscopy (alive or fixed) to determine the gene expression.]
The protocol for fluoresence microscopy can be found at:https://www.protocols.io/view/transfected-naegleria-fluorescence-microscopy-hpvb5n6
TITLE: Bioluminescence Imaging
AUTHORS: Santiago Unda, Michael G. Kaplitt

[DESCRIPTION]
This protocol describes a bioluminescence stepwise imaging protocol in mice with AAV-luciferase
expression in the vagal afferents using the Ivis Lumina series III imaging system. 

[STEPS]
SECTION: Preparation and In vivo imaging
1. Induce anesthesia with isoflurane 2-3%. 
SECTION: Preparation and In vivo imaging
2. Shave the neck, thoracic and abdominal area.
SECTION: Preparation and In vivo imaging
3. Prepare D-luciferin potassium salt (Xenogen) stock prepared in phosphate buffered saline (PBS) at 15mg/mL.
SECTION: Software protocol and analysis
6. Living imaging software (Ivis imaging) was configured to luminescent imaging mode with an exposure time of   and 5-minute delay.

SECTION: Preparation and In vivo imaging
4. Inject D-luciferin solution (150mg/kg) intraperitoneal.
SECTION: Preparation and In vivo imaging
5. Image mice in supine position at 5-, 10- and 15-minutes post-luciferin injection.
SECTION: Software protocol and analysis
7. Post-acquisition, the total flux of photons/radiance per second (s) in each pixel in the ROI area, in a square
centimeter (cm2) of the tissue. This data is then multiplied by one steradian (sr), which result in average radiance displayed as (photons/s/cm2/sr).
TITLE: Crystallization of Zika virus NS3 helicase 
AUTHORS: Andre Schutzer de Godoy, Peter Marples, Lizbé Koekemoer

[DESCRIPTION]
The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
43.2 µL5 mg/mL  
4.8 mL 
4.8 mL  seeds, dilution 1:100

SECTION: Crystallization experiment
5. Dispense 50 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 28615 mg/mL    to each lens using the SPT mosquito.
Dispense  100 2861 to each lens using the SPT mosquito.
Dispense  50 2861 to each lens using the SPT mosquito.

Drop ratio: 3:2:1 ratio (150 nL   : 100 nL reservoir solution: 50 nL seeds)
Final drop volume: 300 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.12 Molarity (M)  NPS Mix
0.1 Molarity (M)  MES pH 6.5 
33% Prcipitant Mix 4
Stock solutions used:
1 Molarity (M)  NPS Mix (consisting of 0.3 M Sodium phosphate dibasic dihydrate, 0.3 M Ammonium sulphate, and 0.3 M Sodium nitrate from Molecular Dimensions)
1 Molarity (M)  MES (Molecular Dimensions)
Precipitant Mix 4 (11% MPD, 11% PEG 1,000, and 11% PEG 3,350 from Sigma Aldrich)

 

SECTION: Crystallization experiment
7. 
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 100 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.62
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 3.80e+12
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Previous research in spatial cognition has often relied on simple spatial tasks in static<br />environments in order to draw inferences regarding navigation performance. These tasks<br />are typically divided into categories (e.g., egocentric or allocentric) that reflect different<br />two-systems theories. Unfortunately, this two-systems approach has been insufficient for<br />reliably predicting navigation performance in virtual reality (VR). In the present<br />experiment, participants were asked to learn and navigate towards goal locations in a<br />virtual city and then perform eight simple spatial tasks in a separate environment.<br />These eight tasks were organised along four orthogonal dimensions (static/dynamic,<br />perceived/remembered, egocentric/allocentric, and distance/direction). We employed<br />confirmatory and exploratory analyses in order to assess the relationship between<br />navigation performance and performances on these simple tasks. We provide evidence<br />that a dynamic task (i.e., intercepting a moving object) is capable of predicting<br />navigation performance in a familiar virtual environment better than several categories<br />of static tasks. These results have important implications for studies on navigation in<br />VR that tend to over-emphasise the role of spatial memory. Given that our dynamic<br />tasks required efficient interaction with the human interface device (HID), they were<br />more closely aligned with the perceptuomotor processes associated with locomotion<br />than wayfinding. In the future, researchers should consider training participants on<br />HIDs using a dynamic task prior to conducting a navigation experiment. Performances<br />on dynamic tasks should also be assessed in order to avoid confounding skill with an<br />HID and spatial knowledge acquisition.</p>

[GUIDELINES]
<p>This protocol was written for experiment instructors in the VR study  "Evaluation of a Conceptual Framework for Predicting Navigation Performance in Virtual Reality". It is meant for use in a CAVE system with 3 screens and Vizard software.</p>

[STEPS]
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TITLE: Simple Step to Increase Gibson Assembly Efficiency 
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Gibson assembly solutoin is extremely toxic to some bacteria cells. </div><div class = "text-block">Doing a solution change after assembly and before transforming the bacteria will significantly improve bacteria survival. This increases 10-100x the amount of colonies.</div></div>

[STEPS]
?. [Do Normal Assembly]
Do a normal Gibson assembly. Incubate in the PCR machine 60 min @ 50C
?. [Do Normal Assembly]
Remove the assemblies. Do a PCR quickchange/ gel extract column. This will remove the Gibson Assembly solution but keep the 100ng (or however much) of DNA.Elute in 15u -20ull TE or water
?. [Do Normal Assembly]
Take the 15-20ul assembled plasmid and heat shock your bacteria. 30 second heat shock. 2 min ice. Add 200ul media and shake for 45 min. Carb can be plated immediatly. Plate