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

[DESCRIPTION]
This protocol is based on Schleper et al. (1992) as modified by Stedman et al. (2003).<br /><br />This is a protocol from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors: </strong>Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<br /><br />Generally this method is successful only if there is an indication for the presence of virus, for instance a halo on a lawn. Even when halos are formed, finding viruses by TEM can be challenging; often supernatants are concentrated 10- through 1000-fold by ultrafiltration or ultracentrifugation (Rice et al. 2001).<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bwuc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bwuc2xe.png" data-ofn="MAVE ch 6 fig 2.png" /><br /><strong>Fig. 2:</strong> TEM of <em>Sulfolobus</em> viruses and VLPs. <br />(A) <em>Sulfolobus</em> spindle-shaped virus SSV-I2 particles. <br />(B) <em>Sulfolobus</em> turreted icosahedral virus (STIV). <br />(C) Three different VLPs from an enrichment culture from Amphitheater Springs, Yellowstone National Park, USA. Note end of a <em>Sulfolobus islandicus</em> rod-shaped virus (SIRV)-like particle in upper right of image). <br />(D) Virus-like particles from Amphitheater Springs. All scale bars 200 nm. Negative stain with uranyl acetate.<em> <br /></em>

[STEPS]
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TITLE: Full plate &amp; spot test plaque assays + PFU/mL calc. - aerobic bacteria
AUTHORS: Torben  Sølbeck Rasmussen

[DESCRIPTION]
Protocol for plaque assays - either a full plate plaque assay that are more laborious but with high accuracy, or a spot test plaque assay that are for higher throughput but with less accuracy.

[STEPS]
SECTION: Initial preparation of bacterial culture
1. Spread bacterial culture on an agar plate and incubate at required temperature until clear colonies appear.
SECTION: Initial preparation of bacterial culture
2. Inoculate with a single colony of the bacteria to prepared media, incubate at required temperature, and wait until exponential phase has been reached. Preferably an OD600 between 0.2 – 0.5. Then continue to either Full plate plaque assay or spot test plaque assay - depending on the purpose
SECTION: Full plate plaque assay
3. Prepare a 10-fold dilution series of phages in SM-buffer.
SECTION: Full plate plaque assay
4. Prepare test tubes with 3-4 mL melted soft agar cooled down to 50°C in a heating block
SECTION: Full plate plaque assay
5. Add 300 µL of bacterial culture
SECTION: Full plate plaque assay
6. Add 50 µL of 1M CaCl2 and 50 µL of 1M MgCl2
SECTION: Full plate plaque assay
7. Add 100µL of the 10-fold dilution series made of the phage lysate.
SECTION: Full plate plaque assay
8. Do not vortex, but mix by shaking carefully
SECTION: Full plate plaque assay
9. Immediately after pour on an agar plate. Let it solidify for 15-20 min or until the lid is clear for condensate water
SECTION: Full plate plaque assay
10. Incubate plates at 37°C or other required temperatures.
SECTION: Full plate plaque assay
11. Count the plaques and choose the plates with 30-300 plaques to calculate PFU/mL
SECTION: Full plate plaque assay
12. (PFU / 0.1 mL) * (1/dilution) = PFU/mL.
SECTION: Spot test plaque assay
13. Prepare a 10-fold dilution series of phages in SM-buffer.
SECTION: Spot test plaque assay
14. Clearly mark on each plate the area for each dilution.
SECTION: Spot test plaque assay
15. Prepare test tubes with 3-4 mL melted soft agar cooled down to 50°C in a heating block
SECTION: Spot test plaque assay
16. Add 300 µL of bacterial culture
SECTION: Spot test plaque assay
17. Add 50 µL of 1M CaCl2 and 50 µL of 1M MgCl2
SECTION: Spot test plaque assay
18. Immediately after pour on an agar plate. Let it solidify for 15-20 min or until the lid is clear for condensate water
SECTION: Spot test plaque assay
19. Deposit 10 µL of phage suspension on top of the marked area. The lid can be slightly opened to speed up the drying.
SECTION: Spot test plaque assay
20. Dry plates until the phage solution have evaporated - may take 30-90 min. 
SECTION: Spot test plaque assay
21. Incubate plates at 37°C or other required temperatures.
SECTION: Spot test plaque assay
22. Count the plaques lysis and choose the plates with 10-50 plaques to calculate PFU/mL
SECTION: Spot test plaque assay
23. (PFU / 0.01 mL) * (1/dilution) = PFU/mL
TITLE: Tissue Fixation Preparation
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes fixation of tissue in preparation for pathology review to assess tissue quality.  
Prior to performing molecular analyses, it is critical to have experienced pathologist confirm the tissue of interest is captured, the region of interest is represented and there are no abnormalities.  Fixed tissue is preferred for pathologist review as it prevents autolysis and degradation of the tissue, cells and cellular components so they can be observed both anatomically and microscopically in their most natural state following sectioning.  Various reagents can be used for fixation.  Formaldehyde is a water-based solution and is the most commonly used chemical fixative.  Paraformaldehyde has been found to be a preferred fixative for the ovary as it maintains oocyte and follicular morphology which is often disrupted with other fixatives.

[STEPS]
SECTION: 4% PFA Tissue Fixation of Ovaries
1. Place each pre-weighed piece of tissue in either a 1.5mL Eppendorf tube or 1.5mL cryo tube.
SECTION: 4% PFA Tissue Fixation of Ovaries
2. Keep PFA tubes on ice. Transfer tissue specimen into a tube and refrigerate at 4 °C for  1440 min .
SECTION: 4% PFA Tissue Fixation of Ovaries
3. After 1440 min, transfer tissue into 4% sucrose/PBS solution and incubate for 1440 min at 4 °C.

SECTION: 4% PFA Tissue Fixation of Ovaries
4. After 1440 min, transfer tissue specimen to 70% EtOH (freshly made) and store at 4 °C until ready for paraffin embedding.
SECTION: 4% PFA Tissue Fixation of Ovaries
5. Proceed with paraffin embedding of samples within 3 weeks.
SECTION: 4% PFA Tissue Fixation of Ovaries
6. Paraffin embedded tissue can now be used for H&E Pathology Review.
SECTION: Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
7. Place each pre-weighed piece of tissue in either a 1.5mL Eppendorf tube or 1.5mL cryo tube.
SECTION: Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
8. Place tissue specimen in a prepped and labeled tube. Keep at room temperature for 1440 min.
SECTION: Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
9. After 1440 min, transfer tissue specimen to 70% EtOH (freshly made) and store at 4 °C until ready for paraffin embedding.
SECTION: Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
10. Proceed with paraffin embedding of samples within 3 weeks.
SECTION: Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
11. Paraffin embedded tissue can now be used for H&E Pathology Review.
TITLE: Viral-mediated short-hairpin RNA knockdown
AUTHORS: Chuyu Chen, Loukia Parisiadou

[DESCRIPTION]
Stereotaxic injection of viral vectors for gene knockdown experiments : The AAV vectors for knocking down the gene of interest were custom-made with Vector Biolabs using AAV-GFP-U6 vector for AAV1 packaging (Cat no, 7040, Vector Biolabs). They were then unilaterally injected into the striatum of mice as follows.

[STEPS]
SECTION: Just before the procedure
1. The following preparatory steps are followed: 
SECTION: Just before the procedure
1.1. Weigh the animals
SECTION: Just before the procedure
1.2. Calibrate the David Kopf frame based on the manufacturer’s instructions. 
SECTION: Just before the procedure
1.3. Clean the Hamilton Syringe with 70% ethanol and distilled water. 
SECTION: Just before the procedure
1.4. Shave the fur off the top of the animal’s skull.
SECTION: Viral injections
2. Anesthetize the animal with 3-4% Isoflurane. Monitor closely the animal until it reaches anesthesia (typically 5–10 min) and does not respond to a light pinch to the hind paw or tail.
SECTION: Viral injections
3. Place the animal in the stereotaxic instrument by carefully placing one ear bar in the ear canal, securing the bar, and holding the animal in place as the other ear bar is placed and secured. 
4. Secure the mouth in the incisor adapter of the stereotaxic instrument. It is critical to ensure that the airway is not blocked. 
5. Cover the anesthetized animal’s eyes with sterile ocular lubricant to keep them moist during the surgery.
6. Clean the skin’s surface with alcohol and Iodine prep wipes.
7. Make a midline incision to the skin’s surface’s skull with small surgical scissors or a scalpel.

8. Clean the skull with cotton swabs.
9. Identify the coordinates relative to bregma:
four sites in the striatum 
anterior-posterior 0.8 mm,
mediolateral 2.4 mm 
dorsoventral –2.8 and −3.6 mm 
anterior-posterior 0.2 mm 
mediolateral 1.8 mm, 
dorsoventral –2.8 and −3.6 mm relative to bregma
10. Load the viral solution with Hamilton 
11. Find bregma with the syringe needle and zero everything in the stereotactic frame 
12. Determine your coordinates. Deliver virus by 100 nl/min speed (500-700 nl of AAV1-GFP-U6-shPKARIIβ or AAV1-GFP-U6-sh control viruses (5x10^12 genome copies/ml)
13. After injections, wait 10 minutes to allow the virus to diffuse 
14. Remove the syringe very carefully and close the surgical sutures

15. Make a subcutaneous injection of Meloxicam right after the surgery and up to 48 hours.
16. Move the animal to the cage on the heat pad and monitor carefully its behavior. 
17. Perform behavioral and biochemical experiments 4 weeks post operation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>DNA isolation from gram negative bacteria with chemical SDS lysis (Phenol) for sequencing on Nanopore. Fast low input cell culture (5ml) with max output (2-3 µg) and longer reads ( 30kb+) .</p>

[BEFORE_START]
<p>Prepare buffer ( taken from http://bit.ly/2FfjomS @Josh Quick)</p>
<p> </p>
<p>TLB:</p>
<p> </p>
<p>10 mM Tris-Cl, pH 8.0</p>
<p>25 mM EDTA, pH 8.0</p>
<p>0.5% (w/v) SDS</p>
<p>20 µg/ml Qiagen RNase A (add fresh just before use)</p>

[STEPS]
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TITLE: KAPP-Sen TMC: Dissociation of Pancreatic Islets (non-recovered) Protocol
AUTHORS: Juliana Alcoforado Diniz, Dylan Baker, Jessica Garofalo, Paul Robson

[DESCRIPTION]
The dispersed samples were shipped cold from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets were dissociated as follows.




[STEPS]
SECTION: Abstract
1. The dispersed samples were shipped cold from PRODOLABS.
Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets
were dissociated as follows.
SECTION: Cell Dissociation with Accutase
2. NOTE: Before beginning cell dissociation coat all the materials (pipettes, tubes, etc.) with PIM-S001GMP media to prevent sticking. 
Transfer cell suspension of pure islets to a new 50ml tube. Use additional media to rinse original container.
Centrifuge at room temperature 130g for 5 mins. 
Aspirate the supernatant and add media to the appropriate concentration of 1,000 islets/1 ml.               Ex: If receiving 7,000 islets, use 7 ml media.
Add half of the islets (recovery) to the coated flask and put to incubate at 37C.
To the other half of the islets (no recovery), add media to 10 ml and centrifuge then aspirate carefully.
Add accutase at a concentration of 1 ml/1,000 islets. Mix with pipette and incubate at 37C. Coat pipette tip and use to mix every 2 mins, checking at 6 mins. 
Add CMRL 1066 (Cat. 11530037) to approximately 9 ml media/1 ml accutase then centrifuge at 230 g.
Aspirate supernatant and resuspend in 1.5 ml of CMRL
Filter through a 40 µm Flowmi.
Count cells using AO/PI (acridine orage/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
Proceed to cell fixation.
SECTION: Fixation of Cells &amp; Nuclei for Chromium Fixed RNA Profiling
3. Cells were fixated prior to scRNAseq according to https://dx.doi.org/10.17504/protocols.io.[...] /v1
TITLE: Sacrificing Rat
AUTHORS: Shaina Robbins, Alison Moss

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the standard procedure we use when sacrificing our Sprague-Dawley rats.</div></div>

[STEPS]
?. [Sacrificing Rat]
Expose animal to 5% isoflurane by inhilation until unconsiousSacrifice animal by means of decapitationHarvest organs per experimental design
TITLE: Striatal dopamine measurement through HPLC
AUTHORS: Pranay  Srivastava, Waijiao Cai, Xiqun Chen

[DESCRIPTION]
Protocol for striatal dopamine measurement in mouse brain

[STEPS]
1. Weigh the striatal tissue 
Note - make sure to keep it frozen – can keep it on dry ice for the process
2. Add 20x PE buffer supplemented with an internal standard solution to tissue. 
(eg. 10 mg sample tissue would receive 200 uL of PE solution)

    
3. Use a Teflon pestle to homogenize the tissue into the solution by crushing it against the walls of the tube. 
Note - Do not sonicate as it may oxidize our analyte)

Note - Keep homogenized samples on ice until centrifugation

Note - If using a hand homogenizer, note that the machine produces heat during prolonged use which can degrade the analytes. Allow time to cool between samples.

Alternatively, can use Dounce homogenizers
4. Centrifuge at 14000 rpm, 15 min, 4 °C

5. Remove supernatant and transfer to Costar Spinx (#8161) 0.22 - MCA filter tube 
Centrifuge for 5 minat 14.000 rpm, 5 min, 4 °C or until all liquid has passed through the filter.

Note - Filters have a max volume of 500uL, and may have to perform multiple spins/samples if the supernatant volume is >500ul.
6. The resulting solution can be run on HPLC or stored at -80 °C.

7. Running a standard - HPLC

Perform serial dilution of the stocks as follows to create the standard curve.

 
  


 
 
 
8. Load at least 50 uL into each vial to run the standard curve in triplicate (on HPLC).

Run the samples
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 12">
<div class="layoutArea">
<div class="column">The steps for preparing the lysate are different depending on the starting material. Please ensure you follow the correct procedure for your starting material (see the section <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Disrupting and Homogenizing Starting Materials</a> and the different lysate preparation protocols <a href="https://www.protocols.io/view/isolate-ii-biofluids-rna-kit-f5kbq4w" target="_blank">here</a>). The subsequent steps detailed in <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw" target="_blank">Genomic DNA removal and total RNA purification</a> are the same in all cases.</div>
<div class="column"><br />
<ul>
<li>
<p>This protocol is designed for the isolation of all sizes of RNA (including miRNA) from a wide variety of biofluids such as saliva, urine, semen, CSF and blood. We recommend the use of non-coagulated blood samples (including fresh and anticoagulant treated blood).</p>
</li>
<li>
<p>Follow this protocol for isolating viral RNA from biofluids.</p>
</li>
<li>See <a href="https://www.protocols.io/view/support-protocol-total-rna-purification-from-plasm-f5hbq36" target="_blank">here</a> for isolating total RNA or viral RNA from plasma or serum.</li>
</ul>
</div>
</div>
</div>

[BEFORE_START]
<div class="page" title="Page 12">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>Ensure Lysis Buffer RX is prepared (see guidelines).</p>
</li>
<li>
<p>Ensure that all solutions are at room temperature before use.</p>
</li>
<li>
<p>Two types of spin columns are provided with this kit: the <strong>Genomic DNA Removal Column</strong></p>
<p>(blue ring) and the <strong>RNA Column</strong> (black ring). Ensure the correct column is used for each</p>
<p>step of the procedure.</p>
</li>
<li>
<p>All centrifugation steps are carried out in a benchtop microcentrifuge at 14,000 x g except</p>
<p>where noted. Perform all centrifugation steps at room temperature.</p>
</li>
<li>
<p>It is important to work quickly during this procedure.</p>
</li>
</ul>
</div>
</div>
</div>

[GUIDELINES]
<ul>
<li>It is recommended that no more than 100 μL of biofluid is used in order to prevent clogging of the column.</li>
<li>Biofluids derived from all human and animal sources are considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with biofluids.</li>
</ul>
<p> </p>
<p>Please review the Guidelines under <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Genomic DNA removal and total RNA purification from all types of lysate</a> for other important details.</p>
<p> </p>

[STEPS]
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TITLE: The Crick COVID-19 RT-PCR Testing Pipeline
AUTHORS: The Crick COVID-19 Consortium

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Crick COVID-19 Consortium commenced on 19 March 2020 in response to the emerging COVID-19 pandemic in a collaboration between University College London Hospitals, Health Services Laboratory (HSL), the Institute of Cancer Research and the Francis Crick Institute.</div><div class = "text-block">Over 2 weeks, the consortium has set up a high throughput RT-PCR COVID-19 diagnostics assay in an academic environment. The work is conducted at the Francis Crick Institute as an extension of the accredited laboratories at HSL. The aim has been to minimise dependencies on reagents, in short supply globally, to provide resilience to the pipeline and provide a scale-able platform to screen hundreds to thousands of healthcare workers and patients per day.</div><div class = "text-block">The interactive Standard Operating Procedures (SOP) provide in this collection a step-by-step approach to set up a diagnostic pipeline in an academic institute in collaboration with partner laboratories and hospital trusts.</div><div class = "text-block">--</div><div class = "text-block">The members of the Crick COVID-19 Consortium would like to thank Julia Roßmanith, Friedrich-Alexander Universität Erlangen-Nürnberg, for her assistance with the editing of this collection of protocols.</div></div>

[STEPS]
TITLE: Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol explains methods for dephosphorylation of 5´-ends of DNA using rSAP in restriction enzyme reaction (M0371).

[STEPS]
1. Digest 1 µg–5 µg in a 20 μl reaction as follows:

   DNA ≥ 1 μl    Restriction Enzyme Buffer (10X) 2 μl   Restriction Endonuclease 1 μl   H2O, purified to 20 μl**   
 

2. Incubate at 37 °C for 60 min or follow manufacturer’s recommendations.
3. Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 µg of a 3 kb plasmid).
4. Incubate at 37 °C for 30-60 minutes.
5. Stop reaction by heat-inactivation of rSAP and restriction enzyme (follow manufacturer's recommendations).
TITLE: Western blotting
AUTHORS: Marine Houdou, rosanne.wouters, Peter Vangheluwe

[DESCRIPTION]
Protocol to detect proteins via Western Blotting.

[STEPS]
SECTION: Harvesting cells
1. collect the cells by scrapping them with a scrapper in Dulbecco’s Phosphate Buffered saline modified without calcium chloride and magnesium chloride (DPBS)  or, using 0.25% Trypsin-EDTA for which stop enzymatic reaction by adding culture medium.
SECTION: Harvesting cells
2. Centrifuge cell suspensions at 450 g, 4°C for 5 min.

SECTION: Harvesting cells
3. Resuspend cell pellets with DPBS and centrifuge following the same indications as in 2. Repeat once.
SECTION: Harvesting cells
4. Discard supernatants and keep cell pellets on ice.
SECTION: Cell lysis and protein concentration determination
5. Resuspend cell pellets in RIPA buffer (RIPA Lysis and Extraction Buffer supplemented with protease cocktail inhibitors. 
SECTION: Cell lysis and protein concentration determination
6. Vortex 30 s and keep on ice for 30 min.

SECTION: Cell lysis and protein concentration determination
7. Centrifuge at 20,000g, 4°C for 30 min.

SECTION: Cell lysis and protein concentration determination
8. Keep supernatants on ice to proceed with protein concentration determination using the micro-BCA Protein Assay Kit.
SECTION: SDS-PAGE
9. Loading
SECTION: SDS-PAGE
9.1. Mix 20 µg of protein with NuPAGE LDS sample buffer and 5% β-mercaptoethanol final.
SECTION: SDS-PAGE
9.2. Load protein on pre-cast 4-12% Bis-Tris gels. Include at least one lane with a protein ladder.
SECTION: SDS-PAGE
10. Running
SECTION: SDS-PAGE
10.1. Run for 10 minat 100V and 90 minat 110-130V.

SECTION: SDS-PAGE
11. Transfer
SECTION: SDS-PAGE
11.1. Transfer onto PVDF membranes using a liquid transfer and following settings: 100V, 75 min, 4°C.

SECTION: SDS-PAGE
12. Blocking
SECTION: SDS-PAGE
12.1. 60 min Block membranes with blocking buffer (5% milk powder in 1X TBS and 0.1% Tween20 (REF)) for 60 min at room temperature, 19 rpm.

SECTION: SDS-PAGE
13. Primary antibodies
SECTION: SDS-PAGE
13.1. Incubate membrane with primary antibodies in solution (1% bovine serum albumin in 1X TBS-Tween20 (TBS-T) buffer), 60 minat 4°C, 19 rpm.

SECTION: SDS-PAGE
13.2. Wash membrane three times for 5 min in TBS-T, 19 rpm.

SECTION: SDS-PAGE
14. Secondary antibodies
SECTION: SDS-PAGE
14.1. Incubate membrane with peroxidase-conjugated secondary antibodies in solution (1% milk powder in 1X TBS-T) for 60 minat room temperature and, 19 rpm.

SECTION: SDS-PAGE
14.2. Wash membrane five times for5 minin TBS-T, 19 rpm.

SECTION: SDS-PAGE
15. Detection
SECTION: SDS-PAGE
16. Use a chemiluminescence reagent to detect signal and acquire with a Biorad Camera (Vilber Lourmat) and its software (ImageLab).
SECTION: SDS-PAGE
11.2. Scan membrane if necessary.