eamag/protocols-with-steps huggingface dataset:
TITLE: Making Differentiation Media for SH-SY5Y
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This describes how to make differentiation medium for SH-SY5Y using all-</span><span style = "font-style:italic;">trans-</span><span>retinoic acid.</span></div></div>

[STEPS]
?. [Make ATRA Stock Solution]
In a 15 mL centrifuge tube, add  ethanol andautoclaved deionized water. This will make a 95% ethanol solution.
14 ml
0 ml
?. [Make ATRA Stock Solution]
Weigh out ATRA.
5 mg
ATRA is light sensitive, so do this step with the lights off.
?. [Make ATRA Stock Solution]
Transfer of 95% EtOH solution to another 15 mL centrifuge tube.
3330 µl
?. [Make ATRA Stock Solution]
Add the ATRA to the 95% EtOH solution. This makes a 5 mM stock ATRA solution.
5 mg
3330 µl
?. [Make ATRA Stock Solution]
Vortex mix the solution.
?. [Make ATRA Stock Solution]
This solution is used at a rate of stock solution per differentiation media. The final concentration should be 10 μM.
100 µl
50 ml
We most recently made 10 aliquots, each with  . This is to make 10  media batches, each using  .
?. [Make ATRA Stock Solution]
Stock solution can be stored for up to 6 weeks at .
?. [Make l-Glutamine Stock Solution]
Weigh out  l-glutamine.
175 mg
?. [Make l-Glutamine Stock Solution]
Aliquot  NBM into a 15 mL centrifuge tube.
15 ml
?. [Make l-Glutamine Stock Solution]
Add the l-glutamine to the NBM.
?. [Make l-Glutamine Stock Solution]
Vortex mix the solution.
?. [Make l-Glutamine Stock Solution]
Aliquot the solution into aliquots.This solution is 20x. The concentration is 80 mM and the final desired concentration is 4 mM.
1 ml
This protocol makes 10 aliquots of  . Each stock aliquot makes   differentiation medium.
?. [Make l-Glutamine Stock Solution]
Store aliquots at .
?. [Aliquot B-27 Supplement]
Aliquot  batches of 50x B-27 supplement.
600 µl
?. [Aliquot B-27 Supplement]
Each aliquot will make differentiation media.
30 ml
?. [Making the media]
Warm NBM in water bath.
Do not mix the media until you are ready to change the media in a flask. It degrades quickly.
30 ml
?. [Making the media]
Thaw one 50x B-27 supplement aliquot, and one 20x l-glutamine supplement.
?. [Aliquot B-27 Supplement]
Store aliquots at .
?. [Making the media]
In biosafety cabinet, combine the following in a 50 mL tube Nuerobasal Medium50x B-27 Supplement20x l-glutamine Stock Solution500x All-trans-retinoic Acid Stock Solution
29 ml
600 µl
1 ml
60 µl
?. [Making the media]
Sterile filter the differentiation media.
?. [Making the media]
Use immediately.
TITLE: Condor CBD Gummies Reviews (Shocking Exposed 2022): Beware Scam!!
AUTHORS: markhussaion  

[DESCRIPTION]
·Product Name – Condor CBD Gummies
·Composition –Natural Organic compound
·Side-Effects –NA
·Availability – Online
·Rating –★★★★★
·Official Website:- CondorCBDGummies.com

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[STEPS]
TITLE: mRNA Synthesis and Transfection
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains the mRNA synthesis and transfection of lines ND1014, N1, and ND27760 from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [mRNA Synthesis]
Clone coding sequence of Atoh1 into a vector containing the T7 promoter and poly(A) tail for in vitro transcription.
?. [mRNA Synthesis]
Clone coding sequence of Ngn2 into a vector containing the T7 promoter and poly(A) tail for in vitro transcription.
?. [mRNA Synthesis]
Linearize Atoh1 and Ngn2 vectors.
?. [mRNA Synthesis]
Subject linearized Atoh1 and Ngn2 vectors to mRNA synthesis using the HiScribe T7 ARCA mRNA Kit.
?. [mRNA Synthesis]
Purify mRNAs using the MEGAclear Transcription Clean‐Up Kit.
?. [Transfect mRNA]
Transfect using a cationic lipid (e.g. Stem-In).
?. [Transfect mRNA]
For each well of a 12-well plate, incubate  with  in  for .
[mRNA]
[lipid]
[PBS]
?. [Transfect mRNA]
Add DNA:lipid complexes to cells.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Protocol to co-culture individual colonies of different strains (e.g. transformed with different vectors) on a LB agar plate in a distributed and random manner.</span></p>
<p> </p>
<p><span style="font-weight: 400;">This assay has been used for low cost and open source fluorescence imaging (please see https://osf.io/dy6p2/ for further information &amp; data, https://github.com/SynBioUC/FluoPi for code and http://docubricks.com/viewer.jsp? for hardware assembly)</span></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Single cell sequencing preparation in adult Drosophila Brain
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol covers the preparation of adult Drosophila Brains for single cell RNAseq

[STEPS]
SECTION: Fly brain dissection
1. Dissect 20 male and 20 female brains from 10-day-old flies on ice-cold Schneider’s medium with FBS (Gibco, filtered 10% FBS)
SECTION: Fly brain dissection
2. After a quick spin down, remove the supernatant and wash the brains with ice-cold PBS

SECTION: Fly brain dissection
3. Incubate the brain at 25 °C with 300 µl of 0.05% trypsin-EDTA (Fisher Scientific) with continuous pipetting every 5 minutes

SECTION: Fly brain dissection
4. Pass solution containing brain chunks through a 25-gauge needle (25G 5/8), without introducing air bubbles, roughly 50 times
SECTION: Fly brain dissection
5. After dissociation, the solution was through a 10 µm pluri-select cell strainer (Fisher Scientific), and 400 µl of ice-cold Schneider’s medium containing FBS was added to inactivate trypsin
SECTION: Fly brain dissection
6. Centrifuge for 15 min at 600xg and remove the  supernatant without disturbing the pellet

SECTION: Fly brain dissection
7. Suspend pellet in sterile PBS containing 0.04% BSA
SECTION: SIngle-cell quantification
8. Quantify the number of cells and measure viability using an AO-PI reagent (Logos Biosystems)
SECTION: Prepare sequence libraries
9. Encapsulate cells, prepare and sequence libraries using the manufacturer's protocol (10X genomics & Illumina Inc.)
TITLE: Autofluorescence Reduction and Imaging Marmoset NHP Tissue with a TissueFAXS Imaging System 
AUTHORS: Guoping Feng Lab

[STEPS]
?. [Autofluorescence Reduction]
1) Wash samples in 1X PBS for .
?. [Autofluorescence Reduction]
2) Stain samples free floating with  TrueBlack in  Ethanol for
?. [Autofluorescence Reduction]
3) Rinse samples in 1X PBS for .
?. [Autofluorescence Reduction]
4) Mount samples on a slide with ProLong™ Gold Antifade Mountan.
?. [Imaging Samples]
Image samples with a 5x overview scan to determine the tissue regions and boarders.
?. [Imaging Samples]
Switch to 20x and image both slices with DAPI and 488 to determine the location of GFP+ cells.
?. [Imaging Samples]
Create "Defined Regions" around GFP+ cells.
?. [Imaging Samples]
Once these defined regions are created, switch to the 40x water immersion objective and "Force Focus" on multiple points across the tissue to find upper and lower bounds for focusing.
?. [Imaging Samples]
Compute focus points for each defined region -- Options, Focus (1x1) -- and set your upper and lower bounds for imaging.
?. [Imaging Samples]
Image each defined region with  steps so that cells can be reconstructed for morphology.
?. [Imaging Samples]
Export the FOVs for morphology reconstruction.  Be sure to uncheck “Use Stich” as the stitching is done later in the reconstruction pipline.  Save the Z-stack with "Original" and "16-bit" format.
TITLE: Gel Electrophoresis
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol for gel electrophoresis to isolate the DNA fragments from the PCR products.

[STEPS]
1. Set up the required apparatus:
2. Make an agarose gel by following the steps in the "Preparation of Agarose Gel" protocol. Usually, a 1% agarose gel is used. 

 

3. After the agarose gel has solidified in the gel tray, remove the gel tray from the gel caster and place it in a buffer tank with pre-filled 1x TAE buffer.
4. Add the correct DNA ladder and the sample(s) into the wells.
1.1. Secure the gel tray tightly onto the gel caster.                      
1.2. Place an 8-well or 15-well comb into the gel tray.
5. Close the buffer tank with the tank lid, ensuring that the colour of the electrodes on the tank lid is the same as the one on the buffer tank.
6. Run the gel electrophoresis at 110V for 40 minutes, making sure that bubbling occurs when the gel electrophoresis begins.

7. Upon finishing gel electrophoresis, put the agarose gel onto a UV Sample Tray, next, put it into the Gel Doc EZ System, and then run the gel imaging program on the desktop.
8. Save the gel image.
TITLE: HTTM : Illumina libraries
AUTHORS: Antoine Champie, Amélie De Grandmaison

[DESCRIPTION]
Part three of the HTTM protocol. A low-cost and high-throughput Tn-seq protocol. 
This part cover the preparation of Illumina sequencing libraries form genomic DNA. 

[BEFORE_START]
All steps and master mixes need to be kept on ice  as much as possible. Thermocyclers need to be cooled at 4°C before inserting sample plate.  

[STEPS]
SECTION: Libraries
1. Transfer2.5 µL of DNA from the DNA extraction plate to a new PCR plate. 

SECTION: Libraries
2. Prepare a fragmentation master mix with : 

 

SECTION: Libraries
3. Add 1 µL of the fragmentation master mix to each well. 

SECTION: Libraries
4. Incubate in a thermocycler with the following protocol : 
15 minat 37 °C
30 minat 65 °C




SECTION: Libraries
5. Add 1 µLof 4µM Nextera (NxT) adaptors to each well.

SECTION: Libraries
6. Prepare a ligation master mix with : 
 

SECTION: Libraries
7. Add 3.5 µLof ligation master mix to each well.

SECTION: Libraries
8. Incubate in a thermocycler with the following protocol : 
30 minat 20 °C
10 minat 65 °C

SECTION: Libraries
9. Prepare a PCR master mix with : 

 

SECTION: Libraries
10. Add 192 µLof PCR master mix to each well.

SECTION: Libraries
11. Split the PCR reaction into 4 different plates (50µl per plate). 
SECTION: Libraries
12. Incubate each plate in a thermocycler with the following cycles : 
30 sat 98 °C
15 sat 98 °C 
30 sat 72 °C
Repeat from step 2 for 20~25 cycles*
2 min  72 °C






SECTION: Libraries
13. Pool the 4 PCR replicates together in a PCR plate. 
SECTION: Libraries
14. Transfer2 µLof DNA from the pool plate to a new PCR plate.

SECTION: Libraries
15. Add 2 µLof each barcoding primer to the DNA : 
- Nxt_i5_barcoding
- Nxt_i7_barcoding

SECTION: Libraries
16. Prepare a PCR master mix with : 

 
 
 

 
 
SECTION: Libraries
17. Add 44 µL of the PCR master mix to each well of the plate. 

SECTION: Libraries
18. Incubate in a thermocycler using the following protocol : 
30 s at 98 °C
15 s at 98 °C
1 min at 72 °C (no anneal step)
Repeat from step 2 for 5 cycles
2 min at 72 °C






SECTION: Libraries
19. Pool together 2 µL of each sample.

SECTION: Libraries
20. Purify with Ampure XP SPRI beads using a 0.8 ratio. Resuspend with50 µL of molecular grade water.

SECTION: Libraries
21. Proceed with QC and sequencing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>After euthanasia, testicles were obtained, tunica albuginea was removed with sterile forceps, and seminiferous tubules were treated with collagenase for 10 minutes at 37°C (5 mg/ml of collagenase, SIGMA C5138). Using a trans-illumination method, cycles of spermatogenesis were detected. Sections of seminiferous tubules, developing the Golgi-acrosome complex in stages VI to VIII, were selected and cut under stereoscope. The tubules were placed on slides containing 40 μl PBS and extruded with a cover-slip in order to obtain the spermatogenic cells. Spermatogenic cells were fixed and smeared on slides or transferred to microtubes.</p>

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

[DESCRIPTION]
<p>This protocol is adapted from the QIAGEN RNeasy Plant mini kit (Jan 2011). Specific modifications have been made to apply this RNA extraction to tobacco pericarp or fruit tissue.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Hamstring Muscle Architecture In Professional Rugby Union Players: A One Season Prospective Study
AUTHORS: Kevin Cronin, Shane Foley, Sean Cournane, Eamonn Delahunt

[DESCRIPTION]
Hamstring injuries are very common in field sports. Muscle architecture has been suggested as a risk factor for hamstring strain injury. Various medical imaging techniques (Magnetic Resonance Imaging and Ultrasound) have been developed to assess muscle architecture. Ultrasound is often used to assess in vivo hamstring muscle architecture. The relationship player load (match-play, training, strength & conditioning programme) and hamstring muscle architectural characteristics at the mid-season and end of season time points will be evaluated to determine the extent to which extrinsic factors influence hamstring muscle architecture

[STEPS]
1.
TITLE: U Mass - Uric Acid
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.  Serum uric acid levels are affected by alterations in systemic protein and nitrogen metabolism.  Serum uric acid levels are altered in kidney failure and renal complications of diabetes.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Uric acid test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Build ReVisE on Ubuntu 
AUTHORS: Alexey Zhuravlev, Stepan Orlov, Егор Усик, Alexey Kuzin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Current protocol is intended to show how to build visualization system which is called ReVisE. Such system is aimed to provide interactive visualization of large datasets hosted on a remote server or a supercomputer. The installation is described for OS Ubuntu.</div></div>

[STEPS]
?. Make sure the following ReVisE prerequisites are installedcmake >= 3.18.1gitclang >= 9.0 / gcc >= 7.4Qt >= 5.10 including QtSVG, QtWidgetsNode.jsnpmD languageCUDA Toolkit >= 11.1C++ Boost libraries
?. Go to the directory where the ReVisE repository will be downloaded and built.cd
?. Clone ReVisE.git clone https://github.com/deadmorous/revise.git
?. Go to repository directory and set REVISE_ROOT_DIR environment variable to the path where ReVisE was cloned.cd reviseREVISE_ROOT_DIR=$PWD
?. Optionally set up Qt, and probably some library paths, e.g.export QT_SELECT=qt5-11-2export LD_LIBRARY_PATH=$(qmake -query QT_INSTALL_LIBS):$HOME/oss/lib${LD_LIBRARY_PATH:+:${LD_LIBRARY_PATH}}
This step is typically not necessary. One can need it in the case Qt libraries are installed locally, in a way bypassing package manager.
?. Run bootstrap.shMake sure to also build tsv-utils, necessary to process visualization performance log files./bootstrap.sh --with-tsv-utils
?. Build ReVisE with performance measurement enabled by running the build script like this./build.sh -DS3DMM_ENABLE_WORKER_TIME_ESTIMATION=ON
?. Open Linux terminal. To install ReVisE on a remote machine, connect to it with ssh. To do so type the following command:ssh username@hostname
Skip this step if Linux terminal is already opened and the ssh connection to the remote machine is established.
TITLE: Transformation Protocol for BL21(DE3) Competent Cells (C2527H)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This transformation protocol is to be performed directly in the C2527H tubes. (For the C2527I protocol, see here.)

[BEFORE_START]
Perform steps 1–8 in the tube provided.

[GUIDELINES]
Transformation Protocol Variables

Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency. 

Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step. 

Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42°C is optimal. 

Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.



Chemically competent E. coli cells suitable for transformation and protein expression. 

Highlights
Transformation efficiency: 1–5 x 107cfu/µg pUC19 DNA
T7 Expression Strain
Deficient in proteases Lon and OmpT
Resistant to phage T1 (fhuA2)
B Strain
Free of animal products

Genotype
fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS
λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5

[STEPS]
1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 min.
2. Add 1 µL–5 µL containing 1 pg–100 ng to the cell mixture.
3. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 min. Do not mix.
5. Heat shock at exactly 42 °C for exactly 10 s. Do not mix.
6. Place on ice for 5 min. Do not mix.
7. Pipette 950 µL of 42 Room temperature  into the mixture.
8. Place at 37 °C for 60 min. Shake vigorously (250 rpm) or rotate.
9. Warm selection plates to 37 °C.

10. Mix the cells thoroughly by flicking the tube and inverting.
11. Perform several 10-fold serial dilutions in SOC.
12. Spread 50 µL–100 µL of each dilution onto a selection plate and incubate 60 min at 37 °C.
TITLE: encrypting your data
AUTHORS: Alise Ponsero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to encrypt a folder or a file using the command line on a linux/mac system !</div></div>

[STEPS]
?. [encrypt]
In order to encrypt your data, you'll need to zip your folder or file, creating a password protected archive.People without the password will still be able to look at the names of the files in the archive, but won't be able to read the file.Using the command line, navigate to the folder you want to encrypt and run:zip -re output_archive.zip my_folderwith "output_archive" the name of the encrypted archive you want to create and "my_folder" the name of the folder (or file) you want to encrypt.In the prompt, a password will be aseked twice. Make sure to use a strong password (8-10 characters with special characters)
?. [Decrypt]
To decrypt the archive, use the unzip command as usual. A prompt will ask you the password before unzipping the files.unzip output_archive.zip
TITLE:  Preparation of a protein-LA conjugated to horseradish peroxidase by the periodate method.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein LA, a novel hybrid protein, structurally contains 4 of the Ig Fc-binding and 4 of the Ig Fab binding regions on SpA with 4 of kappa light chain-binding sites of protein L. It has a MW of 65 kDa. Protein LA combines the binding properties of the both SpL and SpA and in some cases, give higher binding affinity than either protein alone. The binding of an Ig to SpL does not interfere with binding of another Ig molecule to the SpA domains and vice versa. Protein LA has been shown to bind effectively to immunoglobulins and their fragments from many species of animals [1]. </div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.</span><span style = "font-style:italic;">West Indian Med J</span><span>. 2013;62(1):12-20.</span></div></div>

[STEPS]
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500 µg of staphylococcal protein-A (SpA) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate. On the other hand mix 500 µg of recombinant protein-L with an equal amount (500 micrograms) of the mix of horseradish peroxidase-sodium periodate.
?. The mixtures are incubated for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
?. The preparation (mix of SpA and SpL) is incubated for 90 min at 4°C in the dark with gentle agitation.
?. Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellets is re-suspended in 500 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 200 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The conjugate is then stored at -20°C until further used.
TITLE: Berryhill Project 2018
AUTHORS: Michelle Altemara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish (Danio rerio) are a popular animal model used in a variety of research areas. As with all animal models, husbandry conditions, including environmental parameters, nutrition, and exposure to pathogens can affect research results. Additionly, zebrafish are tolerant of a wide range of environmental parameters, which has led to wide variation in husbandry practices across facilities around the world. The lack of standard conditions across zebrafish laboratories may play a role in irreproducible experiments. Therefore, a summary of these conditions for the year 2018 have been compiled for a project requiring separate space from the Zebrafish Aquaculture Core (ZAC) facility at the University of North Carolina (UNC) Chapel Hill. For this project a new flow-through rack was installed in the Berryhill building.</div><div class = "text-block">Since the project aimes to investigate differences in health status among the fish, all health data has been omitted from this summary.</div></div>

[STEPS]