eamag/protocols-with-steps huggingface dataset:
TITLE: Membrane Tube Assay
AUTHORS: Liv  Jensen

[DESCRIPTION]
This protocol details about the Membrane Tube Assay.

[STEPS]
SECTION: Membrane Tube Assay
1. Passivate flow cell with 1 mg/mL BSA in imaging buffer.

SECTION: Membrane Tube Assay
2. Rinse flow cell with 2 flow cell volumes of imaging buffer.
SECTION: Membrane Tube Assay
3. Mix GUVs with fluorescent protein and add to flow cell, allowing GUVs to settle on the bottom surface of the flow cell.
SECTION: Membrane Tube Assay
4. Add 1 µL of a 1:1000 dilution of silica beads to flow cell.

SECTION: Membrane Tube Assay
5. Trap a bead in the optical trap, bring into contact with a GUV, and retract, forming a membrane tube.
SECTION: Membrane Tube Assay
6. Visualize protein recruitment to membrane tube with confocal microscopy.
TITLE: ASO transfection of iPSC-derived cells
AUTHORS: james.evans

[DESCRIPTION]
A protocol for transfecting iPSC-derived midbrain dopaminergic neurons with antisense oligonucleotides (ASOs). 

[STEPS]
SECTION: ASO transfection of iPSC-derived mDA neurons
1. Make up transfection mix in N2B27. The transfection mix should be 1/5 of the final volume in the well. 
Transfection mix should have:
1. 0.48 % DharmaFECT transfection reagent
2. ASO (adjust concentration depending on cell type, ASO chemical modification, and knockdown required). Calculate final concentration required in the well not in the transfection mix.
Example - 1000 ul final volume in the well with 300 nm of ASO = 200 ul of N2B27 + 0.96 ul of DharmaFect + 3 ul of 100 uM ASO Stock
SECTION: ASO transfection of iPSC-derived mDA neurons
2. Vortex transfection mix and leave at room temperature for 30 minutes. 
SECTION: ASO transfection of iPSC-derived mDA neurons
3. Aspirate media from cells and replace with the transfection mix. After adding the transfection mix add the rest of the media to the cells to make up the final volume.
TITLE: Selection of stable transformants in Ostreococcus tauri, Ostreococcus lucimarinus and Bathycoccus prasinos
AUTHORS: Francois-Yves bouget

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol  describes the selection and growth of stable transformants in semi solid agarose medium. Developped initially for Ostreococcus tauri, it also works for Ostreococcus lucimarinus and Bathycoccus.</div></div>

[STEPS]
?. [Inclusion of cells in semi-solid agarose medium]
Autoclave a solution of 2.1% low melting agarose in H2O. Keep at 65-90 °C in a water bath.
?. [Inclusion of cells in semi-solid agarose medium]
Add 1 ml of LMP agarose to the 9 ml in one of the tubes. Close the tube, and mix gently  by inverting.
?. [Inclusion of cells in semi-solid agarose medium]
Add 0.5 ml of overnight transformed cells (see protocol on transient transformation), quickly mix and gently pour into the plate. pay attention to avoid bubles . Repeat this process for all tubes.
?. [Inclusion of cells in semi-solid agarose medium]
Let the plates dry open in the flowhood for about one hour, so that the agarose solidifies.
?. [Inclusion of cells in semi-solid agarose medium]
Close the plates and transfer them to large square Petri dishes. Add wet paper towel to keep the chamber humid.  Seal the square plates with medical tape. Place the square plates in the incubator for about 10 days.
?. [Inclusion of cells in semi-solid agarose medium]
For each transformation prepare 8 Petri dishes (55 mm diameter) and 8 x 15 ml tubes each containing 9 ml of ASW plus the required selection. (G418 at 1 mg/ml).
?. [Selection of transformants]
Transformant colonies should appear after 10 to 21 days. Pick colonies using with cut-off yellow tips. Suck out the green colony. Take care not to include any cells from neighbouring colonies.
?. [Selection of transformants]
Transfer the cells to 0,2 ml of ASW medium containing the selection (G418), in 96  wells microplate.  Allow cultures to grow for 7 days in the culture incubator.
?. [Selection of transformants]
After one week transfer to 20 ml culture flasks and grow for 7 to 10 days. Stable integration into the genome by random insertion or homologous recombination  can be detected using PCR (see Lozano et al, Plant Journal 2014). When the transgene containsa luciferase reporter, a first screening can be performed directly  by measuring luminescene in a microplate luminometer.
?.
TITLE: Autofluorescence Microscopy Data Acquisition
AUTHORS: Elizabeth Neumann, Heath Patterson, Jamie Allen, Maya Brewer, Mark de Caestecker, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">Obtain autofluorescence microscopy images of tissues.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">An RGB autofluorescence microscopy image of the tissue section that enables registration and correlation of different imaging modalities on a pixel by pixel basis.</div></div>

[STEPS]
?. If sectioned tissue is frozen, return to room temperature (~20oC) within a vacuum desiccator (~30 min), otherwise proceed directly to step 2.
?. Place microscope slide within adapter and insert into the Zeiss AxioScan Slide Scanner
?. Perform coarse focusing of the tissue using:DAPI filter set (ex. 335-383 nm; em. 420-470 nm, blue)  Lamp power (~90%) and moderate exposure times (~150 ms)
?. Perform fine focusing of the tissue using the following to build a focus map:DAPI (blue) GFP (ex. 450-490 nm; em. 500-550, green)DsRed (ex. 538-562 nm; em. 570-640, red)Lamp power (~90%) and moderate exposure times (~150 ms)
?. Define the imaging region that includes the tissue.
?. Acquire autofluorescence image.
?. Export autofluorescence image as an OME-TIF with the following options:"BigTIFF""Use Tiles""Compress""Convert to 8 Bit"Alternatively, other image file types, such as "BigTIFF" and "pngs" can also be useful, depending on the application.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
KAPA Express Extract is a novel thermostable protease and buffer system that allows for the extraction of PCR-ready DNA from various tissue types in as little as 10 min. This protocol uses half the normal reaction mix.

[GUIDELINES]
When running program on PCR machine incubate at 75&ordm;C and then 95&ordm;C consecutively and then cool the reaction products to 4&ordm;C.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Western blot - alpha-synuclein
AUTHORS: Pietro La Vitola, Eva M. Szegö

[DESCRIPTION]
This protocol describes how to detect alpha-synuclein protein  in mouse dorsal medulla oblongata tissue  by western blot 

[STEPS]
SECTION: Sample preparation
1. Tissue homogenization
SECTION: Sample preparation
2. Electrophoresis and transfer

SECTION: Sample preparation
1.1. Homogenise samples in 150 µL ice-cold lysis buffer  (1% Triton X-100 in 0.1 M phosphate buffered saline solution, pH 7.6 , supplemented with protease and phosphatase inhibitors. 

SECTION: Sample preparation
1.2. Centrifuge samples 

SECTION: Sample preparation
1.5. Mix 4 µg samples in Laemmli sample buffer supplemented with 5 % beta-mercaptoethanol.
SECTION: Sample preparation
1.6.  Heat samples 95 °C 5 min 
SECTION: Sample preparation
2.1. Separate samples (4 µg total protein) by polyacrylamide gel electrophoresis using precast Bolt™ 4-20% Bis-Tris, 1.0 mm, Mini Protein Gels at 120 V 80 min or until dye front reaches the bottom of the gel. Run with pre-stained size markers
SECTION: Sample preparation
2.2. Soak nitrocellulose membrane (pore size: 0.2 µm) with Towbin buffer (see materials)
SECTION: Sample preparation
2.3. Soak transfer sandwich components (2 sheets of filter paper and 2 blotting pads) in Towbin transfer buffer and assemble in the transfer cassette in the following order:
cathode plate
1 x blotting pad
1 x filter paper
gel
nitrocellulose membrane
1 x filter paper
1 x pad
Use a roller to remove any air bubbles
SECTION: Sample preparation
2.4. Place cassette in transfer tank and transfer protein onto 0.2 µm nitrocellulose membranes (300 mA,  90 min  in cold Towbin buffer.
SECTION: Sample preparation
2.5. Rinse membranes with TBS 30 s
3.7. Visualize ECL stained membrane using a BioRad ChemiDoc™. 
3.6. Incubate membrane with ECL 30 s
3.5. Wash with TBS-T 5 min
3.4. Incubate membrane with peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG in TBS-T (1:5000 each).   

3.3. Wash with TBS-T  5 min
3.2. Incubate membranes in blocking solution containing mouse anti-α-synuclein (1:1000; RRID:AB_398108) and rabbit anti-β-actin (1:1000; RRID:AB_2305186) 1080 min Room temperature 
3.1. Block non-specific binding sites with blocking solution (TBS-T: TBS containing 2% BSA and 0.05 % Tween-20) for 60 min
3. Immunodetection of protein bands

SECTION: Sample preparation
1.3. Transfer supernatants into pre-cooled test tubes.
SECTION: Sample preparation
1.4. Measure protein concentration (e.g. BCA method).
TITLE: Micromorphological thin section manufacture AMBI Lab, Universidad de La Laguna, Tenerife, Spain
AUTHORS: Lucia Leierer, Caterina Rodríguez, Carolina Mallol

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the manufacture of micromorphological thin sections performed at AMBI Lab, Universidad de La Laguna, Tenerife, Spain.</div></div>

[STEPS]
?. Oven drying of intact sediment blocks at for
?. Impregnating of sample with a 7:3:0.1 ratio of a mixture of  of         polyester resin (Palatal strained resin UN1866, TNK composites),         styrene (Styrene monomer (CAS: 100-42-5) UN2055, TNK composites)         and a catalyzer (Methyl-ethyl-ketone (Luperox, CAS: 78-93-3), TNK composites)
?. After hardening, cutting of the blocks into 1 cm-thick slabs using a Euro-Shatal M31100 radial saw
?. Glueing onto 9 cm x 6 cm glass slides
?. Trimming to 1mm thickness using a Uniprec ATA Brilliant-220 precision cutting machine
?. Grinding to 30 µm thickness using a G&N MPS-RC-Geology grinding machine
TITLE: Protocol for Dephosphorylation of 5′ ends of DNA using Quick CIP (NEB #M0525)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Quick CIP is a heat-labile version of calf intestinal alkaline phosphatase (CIP) purified from a recombinant source.</div></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Rapid and irreversible heat inactivation eliminates unwanted activity</li><li style = "counter-reset:ol0;list-style-type:disc;">Improved storage stability versus native enzyme</li><li style = "counter-reset:ol0;list-style-type:disc;">Faster reaction setup (no supplemental additives like zinc required) and shorter incubation time</li><li style = "counter-reset:ol0;list-style-type:disc;">Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)</li><li style = "counter-reset:ol0;list-style-type:disc;">Less enzyme required (high specific activity), resulting in a lower cost per reaction</li><li style = "counter-reset:ol0;list-style-type:disc;">No need for multiple phosphatases (Quick CIP removes 5′- and 3′- phosphates from DNA, RNA and dNTPs )</li><li style = "counter-reset:ol0;list-style-type:disc;">Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis</li><li style = "counter-reset:ol0;list-style-type:disc;">Recombinant for purity, consistency and value</li></ul></div></div>

[STEPS]
?. Prepare a  reaction as follows:   AB1DNA1 pmol of DNA ends*2CutSmart®&nbsp; Buffer (10X)2 μl3Quick CIP1&nbsp;μl4H2O, purifiedto 20 μl** * Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.** Scale larger reaction volumes proportionally.
20 µl
AB1DNA1 pmol of DNA ends*2CutSmart®&nbsp; Buffer (10X)2 μl3Quick CIP1&nbsp;μl4H2O, purifiedto 20 μl**
?. Incubate at  for .
37 °C
?. Stop reaction by heat-inactivation at  for .
80 °C
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>Prior to following the staining procedure, dilute one (1) part Nuclear Factor Fixation Buffer (4X) with three (3) parts PBS, and dilute one (1) part Nuclear Factor Permeabilization Buffer with nine (9) parts PBS.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Kidney Functional Tissue Unit (FTU) Segmentation
AUTHORS: Nathan Heath Patterson, ellie.l.pingry, Katerina V Djambazova, Melissa Farrow, Raf Van De Plas, Jeff Spraggins

[DESCRIPTION]
This protocol explains how to apply a segmentation model to autofluorescence microscopy images to find kidney functional tissue units (FTUs). Currently the model allows for segmentation of glomeruli, proximal tubules, thick ascending limb, distal tubules, and collecting ducts. 



[STEPS]
SECTION: Apply kidney FTU segmentation to autofluorescence images
2. Create the python environment needed to run wsimap. See instructions at the wsimap GitHub.
SECTION: Apply kidney FTU segmentation to autofluorescence images
1. Configure the segmentation prediction by creating the dataset .yaml file.  Add the path to the saved model and images in the configuration file.
SECTION: Apply kidney FTU segmentation to autofluorescence images
3. Run the model using the command line from the wsimap directory like so:
 


SECTION: Apply kidney FTU segmentation to autofluorescence images
4. Collect .geojson segmentations in output folder specified in .yaml file from Step 2. These can be visualized in QuPath.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
In the late years, the interest in optoelectronic material is attribute to the discovery of electroluminescence in molecular crystals and conducting polymers. Organic materials have received considerable attention due to their application in electronic and optoelectronic devices such as organic thin film transistors, light emitting diodes (OLEDs), solar cells, flexible printed electronics and so on.

[STEPS]
TITLE: Dynabeads™ Cell Separation
AUTHORS: Manuela de las Casas, Laura Sánchez, Claudia Troncone Clemente, Laura Armero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The aim of this protocol is to separate the aptamers that bind to the cells from the ones that don't. The cells used have a histidin tag that attaches to the beads' streptavidin tag. The beads will then pull the cells and the complex will precipitate. Since the aptamers have high specifity and affinity for particular sequences, those able to recognice the cells will bind to them, being part of the complex. The aptamers that don't recognice the cells, won't bind. This protocol enables to separate aptamers based on their affinity using the target cells.</div><div class = "text-block">The Dynabeads His-Tag Isolation and Pulldown from ThermoFisher is an already proven system to separate and isolate proteins. We have automatised the system with the Opentrons to separate cells and it worked succesfully. But if you don't have an Opentrons machine, it is also possible to perform a manual protocol as described here.</div></div>

[STEPS]
?. Thoroughly resuspend the Dynabeads™ magnetic beads in the vial using a pipette or a vortex. This must be done each time the beads are going to be used.
?. Transfer 100 μL (4 mg) Dynabeads™ to a 1.5 mL eppendorf tube. Place the tube in the magnetic rack for 2 min, to ensure separation. Then aspirate and discard the supernatant. Add your cell sample (Sample Co, prepared in 1X Binding/Wash Buffer) to the beads. Mix well.
?. Incubate for 5 min at room temperature in a balance shaker (moving gently). The incubation time may be increased up to 10 min.
?. Place the tube on the magnet rack for 2 min, then remove the supernatant and keep it in a new tube (Sample Cnb).
?. Wash the beads 3 times with 400 μL 1X Binding/Wash Buffer by placing the tube on the magnet for 2 min and remove the supernatant to a new tube. Resuspend the beads thoroughly between each washing step. The supernatant is kept in the same tube for every wash (Sample Cw).
?. Add 100 μL His-Elution Buffer. Incubate the suspension for 5 min at room temperature in the balance shaker (moving gently).
?. Apply on the magnet for 2 min and transfer the supernatant containing the eluted histidine-tagged cell to a clean tube (Sample Cf).
?. Resuspend the remaining beads in 100 μL LB and transfer it to a new tube (Sample Cne).Proceed to cell counting:
?. a. Make serial dilutions of each of the samples until a concentration of 10-4 in LB. Plate 100 μL of the each final                                                 dilution in LB agar + kanamicine plates.b. Count the cells by using a OD relationship.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Step 1: Enrolment of inmates eligible for study participation</strong></p>
<p><strong>Step 2: Study implementation</strong></p>
<ul>
<li>1<sup>st</sup> day of study: Inclusion of participants and questionnaire administration</li>
<li>2<sup>nd</sup> day of study: Preparing the list of participants for the 1<sup>st</sup> sputum sample collection and chest X-ray</li>
<li>3<sup>rd</sup> day of study: Collecting the 1<sup>st</sup> sputum sample and performing the chest X-ray</li>
<li><a target="_blank"></a> 4<sup>th</sup> day of study: Transport of sputum samples to CeDRes and collection of chest X-ray and laboratory test results</li>
<li><a target="_blank"></a> 5<sup>th</sup> day of study: Collecting the 2<sup>nd</sup> sputum sample, performing the HIV test and preparing the samples to be sent to CeDRes</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: U Mass - Insulin clearance
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Insulin clearance test measure systemic clearance of insulin following a bolus injection. Liver accounts for the majority of systemic insulin clearance following secretion from pancreatic β- cells into portal circulation. Hepatic clearance of insulin may be affected by obesity and in other mouse models of altered metabolism. Alterations in insulin clearance may also affect glucose and lipid metabolism.</div></div>

[STEPS]
?. Mice are fasted for 5 hours prior to the start of experiment.
?. Collect plasma sample (20 µl) before the start of experiment (basal-0 min) to measurebasal insulin and glucose levels.
?. Administer intraperitoneal injection of insulin (0.5 or 0.75 unit/kg body weight) using aninsulin syringe.
?. Collect plasma samples (10 µl) at 10, 20, 30, 60, 90 and 120 min for the measurement ofplasma insulin concentrations.
?. For data analysis, plasma insulin levels vs. time after insulin injection are plotted, andarea-under-curve may be calculated to estimate systemic insulin clearance.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Lysis buffer for extraction of DNA from fungal material. Contains ionic and non-ionic detergents. Contains RNAse-A.</p>
<p> </p>
<p>Composition:</p>
<table style="border-collapse: collapse; width: 130pt;" border="0" width="130" cellspacing="0" cellpadding="0">
<tbody>
<tr style="height: 15.0pt;">
<td style="height: 15pt; width: 85px;" height="15">NaCl</td>
<td style="width: 85px; text-align: right;">150mM</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; width: 85px;" height="15">EDTA pH 8.0</td>
<td style="text-align: right; width: 85px;">1 mM</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; width: 85px;" height="15">Tris Base pH 8.0</td>
<td style="text-align: right; width: 85px;">10 mM</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; width: 85px;" height="15">Polyvinylpolypyrrolidone (PVPP)</td>
<td style="text-align: right; width: 85px;">2%</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; text-align: left; width: 85px;" height="15">IGEPAL-CA630</td>
<td class="xl63" style="text-align: right; width: 85px;" align="right">0.5%</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; text-align: left; width: 85px;" height="15">Sodium deoxycholate</td>
<td class="xl63" style="text-align: right; width: 85px;" align="right">0.5%</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; text-align: left; width: 85px;" height="15">SDS</td>
<td class="xl64" style="text-align: right; width: 85px;" align="right">1%</td>
</tr>
<tr style="height: 15.0pt;">
<td style="height: 15pt; text-align: left; width: 85px;" height="15">RNAse-A</td>
<td style="text-align: right; width: 85px;">100 µg/mL</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Calibration Protocol
AUTHORS: Olga Khmelnitsky, Magdalena M Julkowska, Hayley  Sussman

[DESCRIPTION]
This is a quick calibration protocol for the Arduino AAWsmo box. 
Use this protocol at the beginning of every new experiment and as needed. 
This is part of a larger phenotyping project in the Julkowska Lab at the Boyce Thompson Institute in Ithaca NY

[STEPS]
SECTION: Code and Uploading
1. Download the calibrate.ino file from GitHub (insert GitHub link here) to a location in your computer that is easy to access with all other Arduino codes that you will be using. 
SECTION: Code and Uploading
2. Open Arduino IDE and open the calibrate.ino file
 



SECTION: Code and Uploading
3. Select the 'Verify' button in the top left of the screen (indicated by a blue circle with a checkmark in it. This will verify your code to check for any errors without having to upload it to the Arduino)
SECTION: Code and Uploading
4. Connect the Arduino to a USB port on your computer
SECTION: Code and Uploading
4.1. Make sure that the scale is leveled, free from any debris underneath the scale, and empty with nothing on it. (This is because once the USB is connected to the computer the power will be supplied to start up the Arduino)
SECTION: Code and Uploading
5. On the calibrate.ino code, select the 'Upload' button at the top left of the screen, indicated by a right-pointing arrow in a blue circle. Wait for the message at the bottom to read 'Done uploading'.
 

 

SECTION: Code and Uploading
4.2. Change the weight units to those you wish to work with by altering:

Serial.print(" g"); //Change this to your desired units and re-adjust the calibration factor if you like 

In this case, the experiment is optimized for 'grams'. Different weight units might have vastly different calibration factors, adjust as necessary.  

SECTION: Calibration
6. Select two objects with known weights. 
 

SECTION: Calibration
7. Open the 'Serial Monitor" in the upper right-hand corner (indicated by a small magnifying glass in a blue square)
SECTION: Calibration
7.1. Upon opening the serial monitor the code will display this message to guide you through the calibration: 
  

SECTION: Calibration
8. Based on the reading, type in either + or - into the text bar at the top of the window and click 'Send". 

 

SECTION: Calibration
9. When you reach a reading that is true to the actual weight on the scale, note down the calibration factor. 
SECTION: Calibration
10. Place the second weight on the scale and observe the deviation from the true weight and adjust the calibration factor to disperse the variance between the upper limit weight and the lower limit weight. 

 

SECTION: Calibration
11. At the end of the calibration note down the date, calibration factor and the achieved accuracy (how much did the weight vary from the true weight)