eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is based on the study done by two members of the Biological and Agricultural Engineering department at UC Davis which analyzes the ability of infrared spectroscopy to detect fungal infections in almonds. The methods section of this article was adapted to fit the protocols.io format. <br /><br />Source: </p>
<p>“Detection of fungal infection in almond kernels using near-infrared reflectance spectroscopy”. Pei-Shih Liang, David C. Slaughter, Alejandro Ortega-Beltran, Themis J. Michailides. Biosystems Engineering, Volume 137, September 2015, Pages 64–72</p>

[BEFORE_START]
Before starting the protocol it is important to have gathered almond kernel samples from different harvest seasons to better understand the pattern of infection for this specific crop.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Tris-HCl Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Tris-HCl Buffer]
Mix  and indicated volume of hydrochloric acid.  ABCDEFGH1mL of HCl44.241 .438.432.521 .912.25.02pH7.27.47.67.88.28.69.0
pH range:   to  
(a) 0.1 M Tris(hydroxymethyl)aminomethane; 12.1 g L-1 (M.W.: 121.0 g mol-1)(b) 0.1 M Hydrochloric acid
[Tris(hydroxymethyl)aminomethane]
ABCDEFGH1mL of HCl44.241 .438.432.521 .912.25.02pH7.27.47.67.88.28.69.0
?. [Tris-HCl Buffer]
Adjust the final volume to  with deionized water.
200 ml
?. [Tris-HCl Buffer]
Adjust the final pH using a sensitive pH meter.
TITLE: Protein Concentration Determination using Qubit 4 Fluorometer
AUTHORS: Steven J Burgess

[DESCRIPTION]
Procedure for quantification of protein concentration using a Qubit 4 Fluorometer. The procedure follows the manufacturer's instructions, this version is adapted for use with samples that have been extracted in protein extraction buffer as part of processing soybean leaf tissue. 

Consult the manual for further details:  
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_Protein_Assay_UG.pdf

[BEFORE_START]
This protocol assumes protein samples have been resuspended in 1x protein extraction buffer (62.5 mM Tris-HCl (pH 6.8), 2 % SDS (w/v); 10 % glycerol (v/v); 2.5% 2-mercaptoethanol (v/v) ) and the dilution factors were determined in relation to processing of 3 size 7 Humboldt leaf disks resuspended in 450 µL of buffer. Preliminary tests should be made when working with different samples or volumes


[STEPS]
SECTION: Create Working Solution for Analysis
1. Create a working solution of Qubit assay buffer by diluting the reagent 1:200 in the provided buffer.
 

SECTION: Create Sample Dilution for Analysis
2. Dilute sample 1:50 (196 µL dH2O + 4 µL sample)
 



SECTION: Create Sample Dilution for Analysis
3. Add 190 µL of Qubit working solution to a fresh Qubit assay tube, one for each sample to be analyzed (including the three protein standards)

SECTION: Create Sample Dilution for Analysis
4. Add 10 µL of protein standard to the appropriate tube and mix by vortexing 3 ss.

SECTION: Create Sample Dilution for Analysis
5. Add 10 µL of diluted sample to the appropriate tube and mix by vortexing 3 s s.

SECTION: Create Sample Dilution for Analysis
6. Allow samples to incubate at room temperature for 15 min 

SECTION: Create Sample Dilution for Analysis
7. Measure protein sample concentration using the Qubit, following the instructions on the machine (i.e. start by measuring the standard curve).
TITLE: cFos DAB IHC Protocol
AUTHORS: Yueyi Chen, Adam Kimbrough, Olivier George

[STEPS]
?. Day 1
?. Rinse in PBS (if sections have been stored in PBS/Azide)
?. Quench endogenous peroxidase activity:    Incubate sections for 20 minutes in 1% Hydrogen peroxide/ PBS (330µl 30% H2O2 in 10 ml PBS)
?. Rinse in PBS 3 times x 10 minutes each
?. Blocking:    Incubate section at room temperature for a minimum of 60 minutes in PBS/ TX100 (0.3%), BSA (1 mg/ml) and 5% normal donkey serum (Jackson)
?. Antibody incubation:    Fos 1:1000 cell signaling cFos Rabbit Monoclonal #2250    Primary antibody diluent    PBS 0.5%, TWEEN 20, 5% normal donkey serum
?. Day 2
?. Rinse in PBS 3 times x 10 minutes each
?. Rabbit ImmPress reagent undiluted 1-2 hours
?. Rinse in PBS 3 times x 10 minutes
?. For cFos DAB NI    Incubate in Vector DAB substrate kit per kit instructions    For 10 mis use: 4dBuffer, 8dDAB, 4dH2O2, 4d NI
?. Incubate primary antibodies 24 hours at RT degrees with agitation
?. check reaction after 2 minutes to reduce background
?. check full development of cFos and note incubation time
?. rinse PBS 3 times x 10 minutes
?. mount on to coated slides    label experiment date, animal # and code, section #, cFos
TITLE: Accessible Health Sentinal Test Protocol
AUTHORS:  John Warner,  Rebecca Silveston, Delphine Dean

[STEPS]
?. [App Download]
Once the user receives their test kit, they will download the Accessible Health App to their phone through the Apple App Store or Google Play store.
?. [App Download]
The app can be found on the store by searching manually or automatically downloaded by using a QR code on the test kit packaging.
?. [App Download]
User follows the steps on the app to input their user data.
?. [App Download]
User opens the Assay test kit pouch inside the box packaging
?. [App Download]
User puts the tab end of the assay on their tongue for at least 10 seconds
?. [App Download]
If the control line does not appear, the user will put the assay back on their tongue for another 10 seconds to collect more saliva
?. [App Download]
Asssay is placed face up inside the box packaging and the phone is placed ontop (on the shelf in the packaging).
?. [App Download]
User taps the start button and closes the specialized box packaging lid.
?. [App Download]
Once the timer on the app goes off, the user can pick up their phone to see their result displayed.
?. [App Download]
Results are stored locally on the phone and uploaded to a secure cloud database when Wifi or sufficient signal is present.
?. [App Download]
Results are reported to appropriate organizations and agencies (e.g., state health departments) and contact tracing is initiated for positive cases.
?. [App Download]
To confirm user identity, two-factor authentication is required of the user.
?. [App Download]
User starts the app and watches the user tutorial video with audio and closed captioning.
?. [App Download]
User waits 15min for the timer alarm (audible and buzzing).
?. [App Download]
If the assay is not detected by the app, an audible and buzzing alarm sounds to alert the user. The app will show a video detailing common user error problems and how to fix them (placing assay or phone upside down, or leaving the packaging opent). The user will check the positioning of the assay and phone and try again.
TITLE: TissueCyte Specimen Embedding Acrylamide Coembedding
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of a fixed mouse brain specimen bonded to an agarose block for blockface imaging and sectioning using the TissueCyte 1000 system.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: FFPE Tissue Sectioning for CODEX
AUTHORS: Marda Jorgensen, Franchesca Farris

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">FFPE tissues for CODEX® analysis must adhere directly onto Poly-lysine-coated coverslips. Preparation and storage of tissue slices are critical for sample integrity.</div></div>

[STEPS]
?. Prepare a water bath at 40°C and place it next to the Microtome.
?. Prepare a clean, dry surface for laying the coverslips next to the Microtome.
?. Use an aerosol spray to clean coverslips from dust and lint prior to use.
?. Insert a new blade for sectioning each block
?. Section the tissue between 5-10 µm thick.
?. Place the sectioned tissue in the water bath for a few seconds and observe it expanding.
?. When the observed expansion is enough to remove folds and wrinkles from the tissue, using the forceps, quickly place a Poly-lysine-coated coverslip in the water bath and gently move it upward towards the tissue. Doing so, the tissue will lay on the coverslip as this is moved out from the water bath.
?. Put the coverslip on a clean surface with the tissue facing up and let it air-dry overnight.
?. Repeat steps 6-8 for each slice of tissue.
?. When the tissues are dry, place each tissue coverslip in an individual slot of the storage box and cover the storage box with the lid. The box of tissue slices can be kept at 4°C for up to 6 months.
TITLE: Chronic Recoverable Neuropixels in Mice
AUTHORS: Emily A Aery Jones

[DESCRIPTION]
This protocol collection explains how to build a low-cost, lightweight system to implant 1 Neuropixels 1.0 probe or 2 Neuropixels 2.0 probes into mice, record during freely moving behavior, then recover the probes for future use. 

Other chronic recoverable designs:
 single 1.0 probe in mice: Juavinett et al, 2019 (https://elifesciences.org/articles/47188)
 multiple 1.0 probes in rats: Luo & Bondy et al, 2020 (https://elifesciences.org/articles/59716)
 single 2.0 probe in mice and dual or moveable 2.0 probes in rats: van Daal, Aydin, & Michon et al, 2021 (https://www.nature.com/articles/s41596-021-00539-9)
 overview of unpublished designs: Neuropixels 2021 course lecture 3.7 by Yoh Isogai (https://www.youtube.com/watch?v=7cZqYIiGvBQ&list=PLfhWmWntvjl64ti_a-MzHlwqwEU0ZlALb&index=18) 

Advantages of this design:
Lightweight (entire assembly, including headbar, dental cement, and tape, weighs <3g for single 1.0 probe or <4g for dual 2.0 probes)
Allows headfixed or freely moving recordings
Quickly attaches to a headstage holder, which provides LEDs for tracking and a counterweight to encourage running
Position and angle of internal mount on the probe adjustable before gluing, allowing implantation into any brain region, but custom adjustment is not required, allowing the same probe to be re-inserted into a variety of sites
Unlike completely enclosed designs, the shank is uncovered during insertion for better visualization, yet doesn't require delicately surrounding the shank with a glue column
Dual 2.0 probes: probes are independently insertable, so can set custom positions, depths, and angles for each

Disadvantages of this design:
Not suitable for larger animals (lightweight design likely can't withstand larger forces, flex cable remains exposed, moisture known to wick up shank into PCB in rats)
External components are assembled around the probe during surgery rather than during assembly, so surgeries take slightly longer
Less elegant than completely enclosed designs and requires a larger skull surface area for gluing
Dual 2.0 probes: how close together the probes can get is limited by some of the components

Assembly preview:
Single 1.0 probe
 

Dual 2.0 beta probes

 


[STEPS]
TITLE: Modular automated bottom-up proteomic sample preparation for high-throughput applications
AUTHORS: Yan  Chen, Nurgul  Kaplan Lease, Jennifer  Gin, Tad  Ogorzalek, Paul D. Adams, Nathan  Hillson, Christopher J Petzold

[DESCRIPTION]
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.


[BEFORE_START]
For this protocol you will need:
- a Beckman-Coulter Biomek FX liquid handler system with a 96-pod head or similar liquid handler system
- a Beckman-Coulter Biomek NX-S8 or NXP liquid handler system with an 8-pod head or similar liquid handler system
- an Eppendorf 5810R centrifuge with S-4-104 rotor or similar centrifuge

[GUIDELINES]
- A Beckman-Coulter Biomek FX liquid handler system with a 96-pod head is used for the protein extraction and quantification protocols. Alternative liquid handlers can be used with appropriate method development.

- A Beckman-Coulter Biomek NX-S8 or NXP liquid handler system with an 8-pod head is used for the normalization protocol. Alternative liquid handlers can be used with appropriate method development.

- A Molecular Devices Spectramax 250 microplate reader is used for the protein quantification assay measurement.

Notes:
- This protocol is set up to measure the amount of protein in duplicate. 

[STEPS]
TITLE: Retro-orbital injection of AAV in mice
AUTHORS: Miguel Chuapoco

[DESCRIPTION]
This protocol is following:

 






[GUIDELINES]
Please adhere to your institue IACUC guidelines of animal care.

[STEPS]
SECTION: Injection
1. Prepare either virus dilution with PBS prepare. Injection is up to 100 μL per mouse.



SECTION: Injection
2. Take one mouse from the cage into an induction chamber and start isoflurane at 3% with flow rate of ~1L/min
SECTION: Injection
3. After the mouse has slow breathing (~1/s) move the mouse to a nose cone delivery of isoflurane at 1.5% with around the same flow rate.
SECTION: Injection
4. Expose the eye ball with two fingers (above and below) and make sure the you are not pressing on the trachea and the mouse's breathing is regular.
SECTION: Injection
5. Use a 31 G insulin syringe at a 30-degree angle beveled downwards until you hit the bone, retract a bit and slowly inject. See cited paper for more details.
 

 


SECTION: Injection
6. Remove the needle slowly and press the eyelid shut for a few seconds.
SECTION: Injection
7. Prior to recovery, apply 1-2 drops 0.5% ophthalmic Proparacaine to the injected eye
TITLE: Plate Streaking
AUTHORS: Carlos Carlos Goller, Carly Sjogren

[DESCRIPTION]
Overview and Goals
Streaking bacteria on agar plates allows scientists to obtain isolated colonies of bacteria. An isolated colony provides up to 10^8 bacteria cells that are genetically identical. This is why we call them clones or refer to them as colonies. The medium we chose to streak out bacteria must provide the nutrients required for prolific organismal growth, and not all bacteria require the same nutrients. There are a variety of types of media and we will use Tryptic Soy Agar (TSA) plates to grow your bacteria, which may or may not support your bacterium's growth. 

After completing this lab you will gain the following lab skills:
Lab safety and proper personal protective equipment (PPE)
Proper use of equipment: bacterial cell culture incubator
Bacterial plate streaking
Bacterial plate incubation

[BEFORE_START]
Preparation
Learn how to perform bacterial plate streaking to obtain isolated colonies.
Watch Streaking for Isolation (3:38 min)
Read this article to learn about ways to streak a plate to obtain isolated colonies: Streak Plate Method- Principle, Types, Methods, Uses (15 min) 
Practice with a pen and paper the “Quadrant Streaking Method.” 
 
 

[STEPS]
SECTION: Procedure for plate streaking
1. Obtain a fresh petri dish containing TSA media.

SECTION: Procedure for plate streaking
2. Label the bottom of the plate (the side containing TSA media)  keeping to the circumference (write on the edge, not in the center) with your bacterial isolate#, your Team#, your initials and today's date. See the diagram below for a visual example. If the surface of the plate is wet, dry it off using a chemical wipe.

SECTION: Procedure for plate streaking
3. Obtain a sterile plastic loop.
SECTION: Procedure for plate streaking
4. Inspect the plate with the bacterial isolate you will characterize.
SECTION: Procedure for plate streaking
5. Using the sterile plastic loop, touch the loop to a single colony on your bacterial source plate.
SECTION: Procedure for plate streaking
6. Streak the bacteria on the loop onto your labeled inoculate plate following the inoculation procedure illustrated above. 
SECTION: Procedure for plate streaking
7. Put your lid back on your plate, turn it upside down (lid facing the benchtop) and bring it to the incubation chamber.
SECTION: Procedure for plate streaking
8. Incubate your bacterial plate overnight with the goal of obtaining single isolated colonies.
This would be the result
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/CviRI-Purification-From-XZ-6E-Virus-Infected-NC64A-er4bd8w" target="_blank">CviRI Purification From XZ-6E Virus Infected NC64A Chlorella</a>.

[STEPS]
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Centrifuge is a classification software used on whole genome shotgun (WGS) reads. It uses a reference database to determine what organisms are found in your sample. This protocol will show you how to access the data on CyVerse and walk you through some questions to help in the analysis of the plots. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Plasmid: NK644 (pAct-mCherryCCTLL)
AUTHORS: David Booth, Nicole King, Heather Middleton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The attached file is the full sequence for a plasmid that uses gene regulatory elements from </span><span style = "font-style:italic;">S. rosetta </span><span>to drive the expression of mCherry with a c-terimnal geranyl-geranylation sequence upon transfection in </span><span style = "font-style:italic;">S. rosetta</span><span>. The mCherry-CCTLL protein that is expressed from this plasmid highlights the plasma membrane of </span><span style = "font-style:italic;">S. rosetta</span><span>. </span></div><div class = "text-block"><span>The plasmid is backbone is pUC19 and can be propagated in </span><span style = "font-style:italic;">E. coli</span><span> with Ampicillin selection. </span></div></div>

[STEPS]
?.
TITLE: Analyzing SARS-CoV-2 Spike Protein Expression in E. coli
AUTHORS: Harley King

[STEPS]
?. Discuss preparation and expression of spike-SUMO fusion protein in E. coli.
?. Use Benchling to calculate sizes of spike-SUMO and RBD-SUMO.
?. Are the proteins from Step 2 seen on Coomassie stain of SDS-PAGE?
TITLE: Purification of PBMCs
AUTHORS: Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the steps and methods of PBMC purification</div></div>

[STEPS]
?. [Ficoll Density Gradient Centrifugation]
Bring Ficoll to .
0 Room temperature
?. [Ficoll Density Gradient Centrifugation]
Clean the blood tubes with 70% ethanol and transfer the blood to a 50mL tube in a BSL2 hood.
?. [Ficoll Density Gradient Centrifugation]
Dilute blood 1:1 in PBS and mix carefully.
?. [Ficoll Density Gradient Centrifugation]
Place  in each of the 50mL conical tubes.
[Ficoll]
?. [Ficoll Density Gradient Centrifugation]
Cover the Ficoll with the blood/PBS mixture using a serological pippette at the lowest speed.
To avoid compromising the purity of the PBMC, Ficoll and blood should not be mixed in any way. It is therefore best to hold the tube at 45º angle and the blood mixture should exit the pipette and run along the wall of the tube very slowly.
?. [Ficoll Density Gradient Centrifugation]
Centrifuge the samples at , selecting acceleration/deceleration rates of 9/1.
Centrifuge: 800 34, 30 min, 20 10
Normally the rotor brakes have to be fully deactivated to effectively prevent phase mixing. It is also very important to strictly adhere to the specified temperatures because temperature differences will change the density rates of the fluids and may have a negative impact on the separation results.
?. [Ficoll Density Gradient Centrifugation]
After completion of the centrifugation, remove the samples from the centrifuge to avoid phase mixing.
?. [Lymphocyte Purification]
Carefully aspirate 2/3 of the top layer (containing the plasma and platelets) using a sterile serological pipette until the interface (containing the mononuclear cells) is within reach.
?. [Lymphocyte Purification]
Using a pipette, aspirate the entire lymphocyte layer while keeping the volume minimal and transfer to a clean tube.
Try to transfer as little Ficoll and supernatant as possible during this step.
?. [Lymphocyte Purification]
Add at least 3 volumes of PBS to the lymphocyte layer and mix very carefully by pipetting up and down.
?. [Lymphocyte Purification]
Centrifuge at  and remove the supernatant.
Centrifuge: 100 34, 10 min, 20 10
?. [Lymphocyte Purification]
Add at least 3 volumes of PBS to the lymphocyte layer and mix very carefully by pipetting up and down.
?. [Lymphocyte Purification]
Centrifuge at  and remove the supernatant.
Centrifuge: 100 34, 10 min, 20 10
?. [Lymphocyte Purification]
Resuspend the cells in FBS + 10% DMSO and cryopreserve in liquid nitrogen.
TITLE: Size selection (Purification)
AUTHORS: Tsu-Chun Hung, Yin-Tse Huang

[DESCRIPTION]
Size selection (Purification)

[STEPS]
1. Calculate the amount of magnetic beads you need and DISTRIBUTE in a new 1.5 mL eppendorf tube (Mix the bottle VIGOROUSLY to make sure it's homogeneous).

For example, if you have 5 samples to clean up, distribute 49.5 μl in a new 1.5 mL eppendorf tube.
   SampleMagnetic beads needMagnetic beads need *1.119 μl9.9 μl218 μl19.8 μl327 μl29.7 μl436 μl39.6 μl545 μl49.5 μl654 μl59.4 μl763 μl69.3 μl872 μl79.2 μl

 

 

3. Mix sample and beads by gently flicking the tube for 5mins 5 min

 

4. Transfer the tube to the 8-strip magnetic rack. 
After all the magnetic beads are arrested, remove the supernatant.
5. Add 200 µL 80% ethanol, flip the magnetic rack around to clean the pellet. 
Wait until all the magnetic beads are arrested, remove the supernatant.

 

6. Repeat the step 5.
7. Flash spin the tube, and put on the magnetic rack. 
Remove ANY trace of liquid residuals using 10 μl pipette.

 

8. Add 10 µL elution buffer. 
Mix gently by flicking the tube and make sure all magnetic beads are dissolved in buffer, then flash spin down the sample.

10. Transfer the tube to the magnetic rack. 
After all the magnetic beads are arrested, collect the 10 µL supernatant to a 200 μl PCR tube or a 8-strip PCR tube.

11. The clean-up sample is now ready for 2' PCR.
9. Incubate the tube in PCR thermal cycler using "37_incubation" program for 10min 10 min .

2. In each 20 µL sample, add 9 µL magnetic beads in.