eamag/protocols-with-steps huggingface dataset:
TITLE: Amplicon clean-up using SPRI beads
AUTHORS: Muhammad Faisal, Olin Silander, Nikki Freed

[STEPS]
?. [Ampure XP bead clean up]
Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
?. [Ampure XP bead clean up]
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add   room temperature SPRI beads to a   reaction.
50 µl
50 µl
?. [Ampure XP bead clean up]
Pulse centrifuge to collect all liquid at the bottom of the tube.
?. [Ampure XP bead clean up]
Incubate for   at room temperature.
?. [Ampure XP bead clean up]
Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. [Ampure XP bead clean up]
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. [Ampure XP bead clean up]
Add  of freshly prepared room-temperature  ethanol to the pellet.
200 µl
?. [Ampure XP bead clean up]
Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.
?. [Ampure XP bead clean up]
and repeat ethanol wash.
?. [Ampure XP bead clean up]
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. [Ampure XP bead clean up]
With the tube lid open incubate for  or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
?. [Ampure XP bead clean up]
Remove the tube from the magnetic rack. Resuspend pellet in  molecular grade water or Elution buffer, mix gently by flicking and incubate for .
10 µl
?. [Ampure XP bead clean up]
Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
?. [Ampure XP bead clean up]
Quantify  product using the Quantus Fluorometer using the ONE dsDNA assay.
1 µl
TITLE: Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping
AUTHORS: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Viviana Gradinaru

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.</div></div>

[STEPS]
TITLE: Adoption and evaluation of the Doha agreement classification system of groin pain in athletes (part 2): a worldwide survey
AUTHORS: Willem Heijboer, Adam Weir, Eamonn Delahunt, Per Hölmich, Anthony G. Schache, Johannes L. Tol, Robert-Jan de Vos, Zarko Vuckovic, Andreas Serner

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

[STEPS]
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Thawing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the thawing of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.


General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
 




[BEFORE_START]
All cell culture plates which are used as feeders to maintain hPSCs are coated for at least 1 hour with autoclaved 0.2% gelatin solution at room temperature. Remove gelatin solution immediately before plating MEF cells. 

0.2% Gelatin Solution
 

 

[STEPS]
1. To recover frozen stocks for MEF expansion, P0 MEF tubes will be thawed in a water bath at  37 °C by gently shaking

2. Thawed cells are transferred into a 15 ml conical tube containing 9 ml pre-warmed MEF medium. Centrifuge the tube at 250 x g, 5 min 

3. Resuspended MEFs (10x106 cells/10 cm plate) are plated in fresh MEF medium and maintained in a humidified incubator (37°C; 5% CO2)
4. 3-4 days after thawing, the cells should be ready for passaging (90-95% confluent)

2.1. MEF medium
TITLE: Freely moving imaging
AUTHORS: Mai-Anh Vu, mwhowe

[DESCRIPTION]
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the steps for imaging in freely-moving mice. Please contact us (mwhowe@bu.edu) if you are interested in using this technique. 

[STEPS]
1. For visualizing fibers in behaving mice custom-built miniscopes (Liberti et al., 2016; Liberti et al., 2017) were positioned above the fiber bundle, focused with an XYZ manipulator, and cemented in place with Metabond (Parkell) under brief isoflurane anesthesia.
1.1. Note: Permanent implantation was used for initial experiments in this study, but baseplate attachment is equally viable.
2. Mice were allowed to recover for 24-48 hrs after attachment, after which imaging was performed in a custom built acrylic arena as mice foraged for Froot Loops (Kellogg’s).
3. The miniscope GRIN objective had an N.A. of 0.45 for high-efficiency light collection and the imaging area was approximately 800 x 600 µm, allowing visualization of a substantial fraction of the fiber bundle.
4. Both behavioral video and fiber signals were acquired at 30Hz using custom MATLAB acquisition software.
5. Neural imaging data were acquired from DAQ and CMOS PCBs from the UCLA Miniscope project (Pnevmatikakis and Giovannucci, 2017).
6. Illumination intensity was delivered by a 470 nm LED (Luxeon Rebel, Lumileds) at an intensity of 0.1-0.25 mW/mm2, controlled by a D-A interface (National Instruments USB-6009) and BuckPuck LED controller (LEDdynamics, Inc).
7. Imaging data were motion-corrected with NormCorre73, corrected for minor background light leak using spatial high-pass filtering, and converted to ∆F/F.
8. Intensity timeseries for each fiber were extracted using manually selected circular ROIs.
9. Locomotor activity was quantified using manual tracking in ImageJ.
TITLE: Purification of NDP52 (untagged)
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes purification of NDP52 (untagged).

[STEPS]
SECTION: Purification of NDP52 (untagged)
1. The human NDP52 cDNA into a pGST2 vector with an N-terminal GST tag followed by
a TEV cleavage site is available from Addgene (RRID: Addgene #187828).
SECTION: Purification of NDP52 (untagged)
2. After the transformation of the pGST2 vector encoding GST-TEV-NDP52 in E. coli Rosetta pLySS cells, grow cells in 2xTY medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C.
SECTION: Purification of NDP52 (untagged)
3. Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 960 min at 18 °C.
SECTION: Purification of NDP52 (untagged)
4. Collect cells by centrifugation and resuspend in lysis buffer.
SECTION: Purification of NDP52 (untagged)
5. Sonicate cell lysates.
SECTION: Purification of NDP52 (untagged)
5.1. Sonicate cell lysates for 30 s. (1/2)

SECTION: Purification of NDP52 (untagged)
5.2. Sonicate cell lysates for 30 s. (1/2)
SECTION: Purification of NDP52 (untagged)
6. Clear lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific).
SECTION: Purification of NDP52 (untagged)
7. Collect the supernatant and incubate with preequilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-NDP52.
SECTION: Purification of NDP52 (untagged)
8. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of NDP52 (untagged)
9. Wash the beads.
SECTION: Purification of NDP52 (untagged)
9.1. Wash the beads twice with wash buffer.
SECTION: Purification of NDP52 (untagged)
9.2. Wash the beads with high salt wash buffer.
SECTION: Purification of NDP52 (untagged)
9.3. Again, wash the beads twice with wash buffer.
SECTION: Purification of NDP52 (untagged)
10. Incubate beads   with TEV protease at 4 °C.

SECTION: Purification of NDP52 (untagged)
11. After the GST tag was cleaved off, filter the protein through a   syringe filter, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).
SECTION: Purification of NDP52 (untagged)
12. Elute proteins with SEC buffer.
SECTION: Purification of NDP52 (untagged)
13. Analyze fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification of NDP52 (untagged)
14. Pool fractions containing purified NDP52.
SECTION: Purification of NDP52 (untagged)
15. After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at -80 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A simple protocol to extract DNA</p>

[BEFORE_START]
<p>If blood is preserved in ethanol, it has to be dried before starting.</p>

[STEPS]
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TITLE:       ELISA for quantification of IL-30 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Monocytes mainly produce IL-30 in response to TLR agonists including bacterial LPS. It acts on monocytes, macrophages, dendritic cells, T and B lymphocytes, natural killer cells, mast cells, and endothelial cells. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-30 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-30 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-30 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-30 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-30 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-30 standard dilutions and the human IL-30 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol contains 4 sections: <br />1. Cesium Chloride Gradient to separate phage from bacteria. Marine viruses around 1.5g/ml<br />2. DNase I treatment of CsCl-purified phage<br />3. Formamide Extraction of DNA from phagE4. <br />4. CTAB</p>

[GUIDELINES]
Needed:<br />
<ul>
<li>Cesium solutions</li>
<li>0.02 µm filter</li>
<li>CsCl</li>
<li>Tubes</li>
<li>Centrifuge @ 22,000 rpm, 4,000 rpm,</li>
<li>0.2 vol chloroform</li>
<li>10-15 µL DNase I</li>
<li>RNase (if needed)</li>
<li>Oak ridge tube</li>
<li>0.1 vol 2M Tris HCL (pH 8.5)</li>
<li>0.2M EDTA</li>
<li>100 µL 0.5M EDTA</li>
<li>10 Fl glycogen</li>
<li>2 vol 100% ethanol</li>
<li>70% ethanol</li>
<li>Sorvall ST 8 Benchtop centrifuge</li>
<li>567 µL TE Buffer</li>
<li>30 µL 10% SDS</li>
<li>3 µL of 20 µg/mL proteinase K</li>
<li>Incubator</li>
<li>100 µL 5M NaCl</li>
<li>80 µL CTAB/NaCl solution</li>
<li>Microcentrifuge</li>
<li>Phenol/Chloroform solution</li>
<li>0.7 vol Isopropanol</li>
<li>50 µL Sigma water</li>
<li>Nanodrop</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Denville LE Agarose is an all purpose agarose for routine nucleic acid electrophoresis of fragments between 500bp-23,000 bp.</p>
<p> </p>
<p>Denville LE Agarose has no detectable DNase or RNase activity.</p>

[GUIDELINES]
<p><strong>Introduction</strong></p>
<p>Denville LE Agarose is an all purpose agarose for routine nucleic acid electrophoresis of fragments between 500bp-23,000 bp. Denville LE Agarose has no detectable DNase or RNase activity.</p>
<p> </p>
<p><strong>Analytical Specifications</strong></p>
<p>Gelling temperature (1.5%)                     36°C ±1.5°C</p>
<p>Melting temperature (1.5%)                   &gt;90°C</p>
<p>Gel strength (1%)                                 &gt;1,200 g/cm²</p>
<p> </p>
<p><strong>Applications </strong></p>
<p><br /> Analytical electrophoresis of DNA and RNA &gt;1,000 bp</p>
<p> Blotting of DNA and RNA </p>
<p> </p>
<p><strong>Suggested Agarose Concentrations</strong></p>
<p> </p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ha2edne.png" data-src="https://s3.amazonaws.com/pr-journal/ha2edne.png" data-ofn="Screen Shot 2016-12-15 at 11.28.02 AM.png" /><br /> </p>
<p><strong>Dye Mobility Table</strong></p>
<p>Migration of double-stranded DNA in relation to Bromophenol Blue (BPB) and Xylene Cyanol (XC) in Denville LE Agarose Gels.</p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ha3edne.png" data-src="https://s3.amazonaws.com/pr-journal/ha3edne.png" data-ofn="Screen Shot 2016-12-15 at 11.28.49 AM.png" /> </p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: ADE 2022 Day 2: PCR
AUTHORS: Tom Little

[DESCRIPTION]
DAY 2: ADE field practical instructions for PCR 

[STEPS]
1. Retrieve your four tubes from the PCR machine.  
2. Add 24ul of  PCR mix to the second tube in your strip. The PCR mix includes buffer, enzyme and primers to amplify the ~650 base fragment of cytochrome oxidase I (COX1) used as a "universal" DNA barcode for animals. Your tubes will now look like this, with the clear liquid (LYS) in tube 1, and a clear liquid (PCR) in tube 2, followed by two empty tubes. Make sure your sequencing identifier is still clearly visible
   

   

3. Put a new yellow tip on a p10 pipette and transfer 1ul of the clear LYS liquid in tube 1 to the PCR mixture in tube 2.  Avoid picking up any lumps of tissue that might still be present, as these will stop the PCR from working. The best way to do this is to set your pipette to 1ul, depress the plunger, then slide the tip of your yellow tip down the side of the tube until it enters the liquid at the meniscus. At this point, gently raise the plunger to pick up 1ul and move it to the PCR mixture in tube 2.  Make sure you are holding the tubes up to eye level, so you can see what is happening.  For example, it is really helpful if you see the 1ul in the tip, and can see that you have actually transferred it to the PCR mix.  Seeing is believing.  Discard the tip when you are done.

    

4. Get a new yellow tip on a P10 or P20, set it to 10ul and gently mix the PCR solution by slowly plunging up and down. 
5. Put the lids on your strip tube and check the labelling is clear. Take your strip tube to the PCR machine and put it in the block.
6. Once the PCR machine is full, the demonstrators will set the PCR amplification cycles running. The PCR machine cycles through 94°C (to denature DNA into single strands), 44°C (to allow the primers to anneal) and 72°C (to allow the DNA polymerase to copy the DNA strand). The PCR proceeds for 30 cycles, and will take about 2.5 hrs, but you do not have to wait for the PCR to finish. 
7. This is a good opportunity to review how PCR works by watching https://www.youtube.com/watch?v=2KoLnIwoZKU
The "primers" mentioned in the video are a key part of what we are doing.  We have added specific primers: two different ~20 nucleotide long bits of DNA that are an exact match to sequences on the COX1 gene, 650bp apart.  This is why our PCR will amplify only the particular 650bp of the COX1 gene that works as a barcode to distinguish species. We had the primers synthesised by a company that specialises in this.  We sent them the sequence of the primers, and they synthesised the primers in an appropriate concentration for PCR
8. Clean up your work area, replacing the tools in their bag, and discarding any used tips. You may now have time to  specimens using the dichotomous keys, but the next session is entirely dedicated to this.
9. Long after you have left, the demonstrators, and Tom and Andrew will retrieve the tubes from the machine.  We will prepare the DNA for sequencing on the Oxford Nanopore Minion.  Time permitting, this will be live streamed to you, details in the next protocol (DNA sequencing)
TITLE:        ELISA for quantification of human immunoglobulin E (IgE) in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>IgE is a monomer. It has a molecular weight of 188 Kd and a serum concentration of 0.00005 mg/mL. It protects against parasites and also binds to high-affinity receptors on mast cells and basophils causing allergic reactions. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK513460/#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a><span style = ":;"></span></div></div>

[STEPS]
?. An anti-human IgE coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human IgE present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-IgE antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-IgE antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of human IgE present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IgE standard dilutions and the human IgE sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Platelets are the primary cellular mediator of thrombosis. This protocol allows for the preparation of washed human platelets. These platelets can then be stimulated or cultured for various purposes. </p>

[BEFORE_START]
<p>Prepare Tyrode's Buffer, or use Tyrode's buffer from a commerical source</p>

[GUIDELINES]
<p>Handle platelets gently at all steps to avoid activating them</p>

[STEPS]
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TITLE: Plant assemble - Plant de novo genome assembly, scaffolding and annotation for genomic studies
AUTHORS: Scott  Ferguson, Ashley  Jones, Justin  Borevitz

[DESCRIPTION]
With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Adapted from Nanodrop ND-1000 User Manual </p>

[STEPS]
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