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

[DESCRIPTION]
<p><em>The EpiQuik™ Total Histone Extraction Kit</em> is a complete set of optimized buffers and reagents for extracting total core histone proteins (H2A, H2B, H3, and H4) from mammalian cells or tissues in a simple 60 minute procedure. The post-translational modifications (PTM) in the histone extracts are kept intact and thus can be used with Epigentek's <a href="http://www.epigentek.com/catalog/histone-modification.php" target="_blank">histone modification</a> assay kits  or in a variety of downstream applications for histone methylation, acetylation, phosphorylation, sumoylation, ubiquitination, citrullination, and ADP-ribosylation  studies.</p>
<ul>
<li>Pre-optimized and simple 1 hour protocol.</li>
<li>Conveniently includes all essential reagents to carry out a histone extraction.</li>
<li>Standardized procedure for reproducible results.</li>
<li>Extracts a high yield of total core histones from as little as 1 mg of tissues.</li>
<li>Post-translational modifications are kept intact.</li>
<li>Does not affect histone modification status or levels.</li>
</ul>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/e6gegr6.gif" alt="Schematic procedure for using the EpiQuik™ Total Histone Extraction Kit" width="250" height="399" data-src="https://s3.amazonaws.com/pr-journal/e6gegr6.gif" data-ofn="op0006schematic.gif" /></p>

[STEPS]
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TITLE: Protein expression using E. coli strain BL21DE3
AUTHORS: Robert Hooftman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In this protocol will be explained how to express proteins using </span><span style = "font-style:italic;">E. coli</span><span> strain BL21DE3 using a plasmid with a T7 promoter. The promoter of T7 polymerase from </span><span style = "font-style:italic;">E. coli </span><span>BL21DE3 has to be induced with IPTG.</span></div></div>

[STEPS]
?. [Day 1]
Prepare an overnight culture of the bacterium encoding for the protein you want to express.
?. [Day 2]
Prepare an Erlenmeyer flask containing LB medium and the right antibiotics
?. [Day 2]
Inoculate the Erlenmeyer flask 1:100 from your overnight culture and grow the bacteria at 37°C and 120 rpm.
?. [Day 2]
Grow the culture to an OD600 of 0.6. This will usually take 2-3 hours.
?. [Day 2]
Take 1 ml of pre-induction sample and cold shock the culture for 30 minutes on ice. Keep the pre-induction sample in the fridge or freezer.
?. [Day 2]
Add IPTG to a final maximum concentration of 1μM. IPTG is used because E. coli BL21DE3 has its T7 polymerase gene under an IPTG sensitive promoter and we have used plasmids with a T7 promoter in our protein expression experiments
?. [Day 2]
Grow the culture overnight (for 18hours) at 20°C and 120 rpm.
?. [Day 3]
Measure the OD600of a 10x diluted post-induction sample. Calculate the “real” optical density by multiplying this number by 10.
?. [Day 3]
Take 1 ml of post-induction sample and spin this, together with the pre-induction sample down for 10 minutes at 4700 g, using a tabletop centrifuge.
?. [Day 3]
Resuspend the pellet of the pre-induction sample in 100μl of MQ. To correct for the difference in OD600 when you load the samples on a protein gel, resuspend the pellet of the post-induction sample in 100μl x("real"OD600 post−induction sample)/(OD600pre−induction sample)
?. [Day 3]
Prepare the samples to be loaded on a protein gel
?. [Day 3]
Run both the pre-induction and post-induction sample on a protein gel
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was designed for collaborators with the Earth Microbiome Project to contribute samples in a standardized fashion. Raw, frozen samples are submitted in individually labeled tubes, 10 aliquots (identical replicates) per sample. Please note that unsolicited samples cannot be accepted.</p>

[GUIDELINES]
<p><strong>Samples for this study will ideally meet the following criteria</strong>:</p>
<ul>
<li>Fresh or frozen sample (no preservatives) in ~10 aliquots</li>
<li>Metadata well characterized</li>
<li>Large proportion of unmapped diversity in at least some samples</li>
<li>Non-human-associated and non-human-perturbed samples will be given priority</li>
</ul>
<p> </p>
<p><strong>Shipping Guidelines </strong></p>
<p> </p>
<p>For <strong>non-soil samples</strong> only (e.g., feces, sediments, biofilms, organic material, water filters), no special permits should be necessary. However, material transfer agreements (MTAs) may be required, and these should be negotiated between the sender and recipiment as early as possible.</p>
<p>  </p>
<p>For <strong>soil samples</strong> only (sediments are not considered soils):</p>
<ul>
<li>Regulated domestic soils must be shipped with the <a href="http://www.earthmicrobiome.org/files/2016/01/Jansson_APHIS_permit.pdf" target="_blank" rel="noopener noreferrer">Permit to Receive Soil</a> issued through the Animal and Plant Health Inspection Service of the United States Department of Agriculture. Collaborators should know whether or not their soils are regulated. If domestic soils are not regulated, the permit is not needed.</li>
<li>The <a href="http://www.earthmicrobiome.org/files/2016/01/PPQ_sticker.pdf" target="_blank" rel="noopener noreferrer">PPQ Form 550 Black/White Label</a> (sticker) must be affixed to the outside of the shipment for any soils shipping from outside the USA, Guam, Hawaii, Puerto Rico, or the US Virgin Islands.</li>
<li>Samples should be shipped with FedEx overnight (or DHL for international shipments). Soil samples must be shipped to the permit holder.</li>
<li>MTAs may also be required by the sending and receiving institutions, and these should be agreed upon as early as possible.</li>
</ul>

[STEPS]
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TITLE: Detection of five bacterial pathogens of rice by multiplex PCR
AUTHORS: Martine Bangratz, Charlotte Tollenaere

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This multiplex PCR protocol is used for the detection of five bacterial pathogens of rice :</span><span style = "font-style:italic;"> Pseudomonas fuscovaginae, Burkholderia glumae and gladioli, Pantoea spp, Xanthomonas oryzae, Sphingomonas spp.</span></div></div>

[STEPS]
?. The amounts described in this protocol are for one sample. Calculate the quantity you need based on your number of sample. Always prepare 10 % more mix.
?. 
?. 
?. Prepare a 2% agarose gel. Load 10µl PCR product and perform the electrophoresis at 100 Volts for 90min
?. Observe the gel under UV light after a ethidium bromide bath
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol details the use of various unix commands commonly used in bioinformatics. Open this protocol inside the virtual machine (details in "Start Instructions") for easy copy, paste of commands into the command line terminal window. </p>

[BEFORE_START]
<p>Before starting, please visit the <a href="http://earthcube.org/group/ecogeo" target="_blank">ECOGEO website </a>for more information on this 'Introduction to Environmental 'Omics' training series. The site contains a pre-packaged virtual machine that can be downloaded and used to run all of the protocols in this protocols.io collection. In addition to the VM, the website contains video and presentations from our initial 'Intro to Env 'Omics' workshop held at the Univ. of Hawai'i at Manoa on 25-26 Jul 2016.</p>
<p> </p>
<p>Please email ‘ecogeo-join@earthcube.org’ to join the ECOGEO listserv for future updates.</p>

[GUIDELINES]
<p><strong>Unix Commands<br /></strong></p>
<table>
<tbody>
<tr>
<td>pwd</td>
<td>rm</td>
<td>grep</td>
<td>tail</td>
<td>install</td>
</tr>
<tr>
<td>ls</td>
<td>'&gt;'</td>
<td>sed</td>
<td>cut</td>
<td> </td>
</tr>
<tr>
<td>cd</td>
<td>cat</td>
<td>nano</td>
<td>top</td>
<td> </td>
</tr>
<tr>
<td>mkdir</td>
<td>'&lt;'</td>
<td>history</td>
<td>screen</td>
<td> </td>
</tr>
<tr>
<td>touch</td>
<td>'|'</td>
<td>$PATH</td>
<td>ssh</td>
<td> </td>
</tr>
<tr>
<td>cp</td>
<td>sort</td>
<td>less</td>
<td>df</td>
<td> </td>
</tr>
<tr>
<td>mv</td>
<td>uniq</td>
<td>head</td>
<td>rsync/scp</td>
<td> </td>
</tr>
</tbody>
</table>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ej6c2w6.png" width="500" data-src="https://s3.amazonaws.com/pr-journal/ej5c2w6.png" data-ofn="ulay.png" /></p>

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TITLE: Protocol for study of the in vitro effect of purified serum anti-anti-anti-SpA antibodies (Ab-3) on cultures of Staphylococcus aureus.
AUTHORS: Angel  Justiz-Vaillant, Patrick E. Akpaka

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This test shows the ability of Ab-3 antibody to inhibit the growth of S. aureus in vitro. The amounts of samples tested were 18 samples from chicks fed with hyper-immune eggs and therefore have the Ab-3, and six samples from chicks fed non-hyperimmune eggs and defenetively do not have the Ab-3. Inhibition of the growth of the bacteria plated out in blood agar was observed only when the Ab-3 was added to said culture media, sugesting that the anti-anti-anti-SpA (Ab-3) bound to the S. aureus and inhibited their ability to proliferate in culture.  On the other hand, bacterial growth was observed when blanks or sera of chicks fed non-hyper-immune eggs were used because these samples lacked the protective Ab-3 [1]. </div><div class = "text-block">Reference</div><div class = "text-block">Justiz Vaillant AA, Akpaka PE, Smikle M, McFarlane-Anderson N (2012) In vitro Inhibition of Staphylococcus aureus isolates by anti-antiIdiotypic antibodies to Staphylococca protein (SpA). J Vaccines Vaccin 3:127. doi:10.4172/2157-7560.1000127.</div></div>

[STEPS]
?. The investigation of the neutralizing ability of purified anti-SpA antibodies is carried out as follows: One ml of brain heart infusion (BHI) broths is placed in 10 sterile test tubes.
?. To each tube is added an equal volume of 10 µl of purified anti-anti-anti-SpA (Ab-3) antibodies in a concentration of 1.25-µg/µl.
?. All samples are tested in duplicate.
?. An inoculum of an ATCC Staphylococcus aureus strain (ATCC #33592) is prepared to 0.5 commercially prepared McFarland scale standard (1=300 x 10^6/ml bacteria concentration). Then, ten µl of this inoculum is added serially from tubes 1 to 10 (prepared for each sample) and the dilutions were reported in this study as a bacterial concentration of 0.1, 0.01, and 0.001.
?. The absorbance value of each tube at 600 nm is measured.
?. A sample from each tube is plated out on Blood agar and incubated overnight at 35oC.
?. Inhibition of the bacterial growth is observed in individual and pooled sera of chicks fed with anti-SpA hyperimmune eggs up to 28 days, 15 days, and eight days period.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol presents a simplified approach for purification of viral strains directly from plaques without the need for preparation of dilutions. Using the 'Tube-free agar overlay' approach viruses are 'picked' directly from source plaques with a toothpick and streaked into molten host lawns in a manner directly analogous to re-streaking based colony purification of bacteria. This approach requires less time and material than dilution based approaches and yields equivalent purification of viruses. Formation of plaques along streaks also offers further confidence that plaques selected for further purification derive from an isolated phage particle rather than a splash containing multiple viruses or a prophage induction event. 

A video demonstrating the approach is available as supplementary material with the original publication (direct link here).

 

 



[BEFORE_START]
Materials to prepare in advance for agar overlay approaches
 
Host culture:This procedure works well with 100uL of overnight host culture for each standard size (90mm) petri dish.

Virus material: 
For Serial Purification of Viruses the starting material may be an agar overlay plate containing a plaque to be purified, or may be a liquid or frozen stock of unknown titer - such as, for example, an archived plaque or plaque-eluates generated using the 'Archiving Plaques' protocol.
 
Bottom agar plates: Prepare 1.0% agar containing media ('bottom agar') in a glass bottle or flask with a stirbar and sterilize, pour 25mL per standard size petri dishes, and allow to solidify. 

Top agar bottle: Prepare 0.3% agar containing media ('0.3% top agar') in a glass bottle with a stirbar and sterilize; though each plate will require only 2mL of top agar, volumes of up to 500mL can be prepared and re-used across multiple days of plating.

[GUIDELINES]
Please also refer to the original publication for a  "Microreview of additional considerations relevant to agar overlays", which highlights methodological aspects that are known or expected to impact the nature, number, and diversity of viruses recovered using agar overlay approaches.

[STEPS]
SECTION: I. Setting up the top agar in a beaker-waterbath
?. 
SECTION: I. Setting up the top agar in a beaker-waterbath
?. 
SECTION: I. Setting up the top agar in a beaker-waterbath
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: II. Streaking for singles: rapid serial purification of viruses from plaques - Streak 1 (Day 1)
?. 
SECTION: III. Streaking for singles: rapid serial purification of viruses from plaques - Streaks 2 &amp; 3 (Days 2 &amp; 3, or later)
?.
TITLE: Introduction to Nanopore
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We want students to have an excellent foundation and knowledge of nanopore sequencing. This lab will provide students with the understanding of how to sequence DNA and process and then analyze the sequence data. The workflow will be covered in detail and includes: sample collection -> sample prep -> library prep -> sequencing -> data collection/ analysis.</div></div>

[STEPS]
TITLE: LAB Agarose Gel Electrophoresis Buffer Recipe
AUTHORS: Brian P Teague

[DESCRIPTION]
A recipe to make lithium acetate / borate agarose gel electrophoresis buffer. This buffer has a lower ionic strength than TAE, but maintains its ability to resolve both small and large bands.

Additionally, it is MUCH less expensive, and if you are short on time (like in a lab course!) you can run the gel at a higher voltage.

From https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011318 via https://bitesizebio.com/25078/faster-even-cooler-dna-gels/

[GUIDELINES]
Use good laboratory practices for measuring weights and volumes.

[STEPS]
SECTION: 25X Buffer Concentrate
1. Weigh out 25.5 g  

 

SECTION: 25X Buffer Concentrate
2. Weigh out 15.5 g  .

SECTION: 25X Buffer Concentrate
3. Mix the lithium acetate and boric acid with deionized water to a final volume of 1 L

SECTION: 25X Buffer Concentrate
4. Pipette a small amount onto a  . Make sure the pH is between 6.5 and 7.0.

SECTION: 1X Buffer
5. Dilute 40 mL of 25X stock solution with 960 mL deionized H2O. Mix well.
TITLE: alpha-Synuclein pre-formed fibrils preparation and stereotactic injection into Mus Musculus brain
AUTHORS: Jean-Louis Parmasad

[DESCRIPTION]
Preparation of seed-competent alpha-Synuclein pre-formed fibrils and subsequent stereotactic injection into Mus Musculus brain as a model of synucleinopathy.

[STEPS]
SECTION: Storage
1. Store alpha-Synuclein fibril preparations at -80 °C with appropriate biohazard precautions and labels until use.

SECTION: PFF preparation (to be done on the same day as experiment)
2. On the day of usage, briefly thaw fibrils on ice.
SECTION: PFF preparation (to be done on the same day as experiment)
3. Transfer required quantity of fibrils to the appropriate tube compatible with your sonicator. Dilute fibrils with normal saline (0.9% NaCl) if required. We used fibrils at a concentration of 5mg/mL.
SECTION: PFF preparation (to be done on the same day as experiment)
4. Program Covaris S220 water bath sonicator to: 
18 W peak incident power;
20% duty factor;
50 cycles per burst;
150 s.
SECTION: Mouse preparation and administration
5. Anesthetize mouse with isoflurane in an enclosed chamber.

SECTION: Mouse preparation and administration
7. Using a 2µl Hamilton Company syringe, take up 1µl of sonicated PFFs or normal saline (0.9% NaCl) using a micro-syringe computerized pump.
SECTION: Mouse preparation and administration
8. Program the micro-syringe computerized pump to deliver 1 μL mouse PFFs (5 mg/mL) or sterile saline (0.9% NaCl) at a rate of 0.1 μL/min (min). 
SECTION: Post-injection
15. Provide appropriate post-surgical care to the mice.
SECTION: Post-injection
16. Wait 6 months for phenotype development, or however long your experiment requires.
SECTION: Post-injection
17. Perform behavioural experiments then collect tissues for biochemical and histological analysis.
SECTION: Mouse preparation and administration
6. Once deeply anesthetized, transfer the mouse to the stereotaxic frame. Insert mouse nose into isoflurane mask.
SECTION: Mouse preparation and administration
9. Align needle to coordinates relative to bregma: −2 mm medial-lateral; +0.2mm antero-posterior.
SECTION: Mouse preparation and administration
12. Begin the administration sequence, following the micro-syringe computerized pump's instructions.
SECTION: Mouse preparation and administration
13. Leave the needle in place for at least 3 min before its slow withdrawal. 
SECTION: Mouse preparation and administration
10. Incise skin. Mark site on skull with a marking pen, and drill 1mm diameter burr hole with handheld drill.
SECTION: Mouse preparation and administration
11. Slowly position the needle to coordinates relative to bregma: −2 mm medial-lateral; +0.2mm antero-posterior and −2.6 mm dorso-ventral (right dorsal striatum).
SECTION: Mouse preparation and administration
14. Close skin with topical skin adhesive.
TITLE: Determination of C4 concentration by the Mancini test.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. An appropriate anti-C4 antiserum (antibody) is poured in the center well of an agar-containing plate.
?. Carefully circular wells are cut and detached from the plates.
?. A series of standards containing known concentrations of C4 are placed in separate wells, while “unknown” human serum samples and control are placed in other wells.
?. A ring of precipitate forms in the area of optimal antigen-antibody concentration, meaning anti-C4 - C4 reaction as the antigen diffuses radially.
?. The diameters of the rings are measured and perceived normally in 48-72 hours.
?. Finally, a standard curve is developed using the ring diameters of the standards versus the concentrations.
?. A curve is then used to plot the concentration of the control and unknown C4 samples.
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 Ichimura, T. 1979 2. Isolation and culture methods of algae. 2.5.B. Freshwater algae [2. Sôrui no bunri to baiyôhô. 2.5.B. Tansui sôrui]. In Methods in Phycological Studies [Sôrui Kenkyûhô], Eds. by Nishizawa, K. &amp; Chihara, M., Kyoritsu Shuppan, Tokyo, p. 294-305 ( in Japanese without English title ).</p>

[STEPS]
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TITLE: Fungal extraction from Xylosandrus compactus
AUTHORS: James Skelton, Craig Bateman, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to extract ambrosia fungi from </span><span style = "font-style:italic;">Xylosandrus compactus</span><span>, the black twig borer. </span></div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Generating Structural Targets
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will provide a basic guide to Generating Structural Targets.</div><div class = "text-block">Note: Steps may vary based upon data.</div></div>

[STEPS]
?. [Introduction]
A few things to keep in mind:This protocol will demonstrate steps that will are most commonly used by the IPA Core for creating a structural target. In this protocol we use T2 data for the example, but keep in mind that structural images can be created by using other types of data and the steps may vary for those images.In the pipeline of processing data from a sample, it's usually a good idea to start by creating your structural image before doing any TORTOISE processing.
?. [Import T2 Data]
First import your T2 DataType:
?. [Import T2 Data]
If you just type: ImportBrukerAnatomical It will bring up your options and explain how they are used i.e. -i, -o, and -c
?. [Import T2 Data]
Type:
?. [Extract T2 Data]
You need to extract the single volume from your T2 data.
?. [Extract T2 Data]
Type:Note: The "3" will vary, based upon your image and the specific volume you want to extract
?. [Rigid Registration]
After your image has been extracted, a rigid registration needs to be made for the image.
?. [Rigid Registration]
Opening your T2 image to MIPAV with the following steps:In terminal type:MIPAV should open automatically.
?. [Introduction]
Log onto the HPC.
?. [Introduction]
In your terminal, go to your directory with raw T2 data.
?. [Extract T2 Data]
View the T2 image in MRtrix, ITK-SNAP, or MIPAV (MIPAV has different instructions which can be seen in step 7). Look at the different echos, and pick the one that most closely resembles the DTI data.To open ITK-SNAP, type:
?. [Rigid Registration]
You may have to "fix" your T2 image if it is not perfectly aligned. For example if your image is tilted, then you should  do a midsagittal line alignment.Go Algorithms>Brain tools>Midsagittal line alignment
?. [Rigid Registration]
Go to file>Open image (A) from diskGo to the directory with data and click on your extracted image.
?. [Rigid Registration]
NOTE: You should see something similar to the image below.     Correction Notes: You may have to "fix" your T2 image if MIPAV has switched the slidding bars for the echos and slices. This problem can be deduced by sliding the top bar to see if the image filters through the slices or if the image just gets darker.If the image gets darker then go to Utilities>4D volume tools>"Swap dims 34." This should fix the swap.You may need to zoom into your image when you first open MIPAV.Simply select the magnifying, shift, and click repeatedly on the imageYou may need to zoom into your image when you first open MIPAV.
?. [Rigid Registration]
Keep in mind that here's other ways of creating a the structural image. For example there's the rigid alignment and landmark methods.