eamag/protocols-with-steps huggingface dataset:
TITLE: Miniscope calcium imaging data acquisition of cortical activity in non-human primates (NHPs)
AUTHORS: Adriana Galvan, Thomas Wichmann

[DESCRIPTION]
The protocol describes head-mounted miniscope calcium imaging data acquisition for NHPs.

[GUIDELINES]
Introduction:

In recent years, calcium imaging has become a standard technique in experiments utilizing rodent experimentation and is now also becoming useful in primate research.  The size of the primate brain precludes the use of this technique to monitor deep brain structures in monkeys, and the relatively low density and substantial brain movement between experiments in NHPs complicate this research.  Nevertheless, the ability to follow an indicator of activity in multiple simultaneously recorded and genetically defined neurons with sub-second time resolution is highly attractive and bound to lead to important scientific insights.  Below, we briefly describe the essential steps in our laboratory’s calcium imaging experiments. 



[STEPS]
SECTION: Procedure
1. The ability to image calcium transients requires prior placement of a GRIN lens assembly (as detailed in a separate protocol dx.doi.org/10.17504/protocols.io.e6nvw15w2lmk/v1).

SECTION: Procedure
2. Calcium imaging starts several weeks after the cortical implantation of the GRIN lens assembly and injection of a virus solution [for example, the authors have used a solution containing a 1:1 mixture of AAV5-TRE-GCAMP6f (9.05x10e13 gc/ml) and AAV5-ThyStTA (5.13x10e13 gc/ml)] 9 to 48 days post lens implantation surgery.  We image calcium transients at least twice weekly.

SECTION: Procedure
6. After completion of the recording session (which may include ‘spontaneous’ periods as well as periods during which the animal is performing a task), save the data for later analysis.  

Subsequently, disconnect the miniscope from the GRIN lens assembly, the cover of the GRIN lens assembly is re-placed and fastened in place, and the head of the animal is released from the restraint. 

SECTION: Procedure
4. Following head fixation, remove the protective cover of the GRIN lens assembly, and connect a miniscope (in our experiments, an nVista3.0 instrument; Inscopix, CA, USA) to the GRIN lens assembly.

SECTION: Procedure
3. At the beginning of the calcium imaging session, the animal is seated in a primate chair, and restrict its head movements (either through the use of a head fixation bolt or through the use of a thermoplastic helmet).

SECTION: Procedure
5. Collect calcium transient data then in the cortical region of interest (for example, the primary motor cortex, M1) using the miniscope.  

When using the Inscopix hardware, imaging and recording control the parameters using their IDAS data acquisition software.  We typically use a frame rate of 10 Hz, an LED power setting of 0.6-0.8, and a sensor gain of 7-8.  
Adjust the electronic focus to a focal plane demonstrating calcium transients.  
Alignment of frames, and, thus, identification of neurons, across several days is helped by also visualizing non-neuron frame elements (such as blood vessels).
TITLE: Mindfulness and Empathy University of Burgos
AUTHORS: Juan Pablo Pizarro, Raquel De la Fuente Anuncibay, Ángela González Barbadillo, González-Bernal, Esther Cubo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mediating effect of mindfulness cognition on the development of empathy in a university context </div><div class = "text-block">We are conducting a study related to Mindfulness and Empathy in the university population in order to</div><div class = "text-block">explore the psychological variables underlying both.</div><div class = "text-block">The study consists of a series of sociodemographic questions and two tests, the duration of which is</div><div class = "text-block">estimated at 10-20 minutes.</div><div class = "text-block">The instruments used were Five Facet Mindfulness Questionnaire (Baer, Smith, Hopkins, Krietemeyer, & Toney, 2006) and Toronto Empathy Questionnaire (Spreng, McKinnon, Mar, & Levine, 2009).</div><div class = "text-block">Participation is completely voluntary. The Research Team undertakes to maintain the anonymity of all</div><div class = "text-block">participants, as well as to treat the information following the ethical rules of research for this type of study,</div><div class = "text-block">so we ask you to answer with the utmost sincerity.</div></div>

[STEPS]
?.
TITLE: Confocal-based bead-binding 
AUTHORS: Dan Tudorica

[DESCRIPTION]
Method of quantifying in vitro binding.

[STEPS]
SECTION: Protein preparation
1.  In order to prepare proteins for a fluorescent bead-binding experiment, you need one protein that can bind a resin (in my case MBP-Rubicon RH domain, which can bind amylose resin) and a fluorescently labelled bait protein (in my case, Rab7-AlexaFluor 647).

To prepare my AlexaFluor 647, used an NHS ester salt, and followed standard manufacturer procedures for labelling, quenching, and cleaning the protein (https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/nhs-ester-oligonucleotide-conjugation)

Measure the degree of labelling of your sample. My degree of labelling was 0.9 fluorophores per Rab7 copy
SECTION: Protein preparation
2. Here, I wanted to assess the impact of phosphorylation on binding. In order to keep the samples as comparable as possible, I fluorescently labelled my Rab7 with 647, then separated the Rab7 into two pools. One pool I phosphorylated using TBK1, the other pool I treated with the same dilution and recovery steps, but omitted the TBK1. See the TBK1 phosphorylation protocol for details.

SECTION: Bead-binding and confocal
3. Washed and prepared a 40 uL suspension of 25% amylose resin in a 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, and 10 mM TCEP buffer. Incubated this resin with 500 nM MBP-RH domain and ~ 4 uM fluorescently-labelled Rab7.

Prepare a negative control consisting of the experimental sample absent the resin-binding partner, in this case MBP-Rubicon RH
SECTION: Bead-binding and confocal
4. Incubate samples on rocker at room temperature for 1 H. 
SECTION: Bead-binding and confocal
5. Collect resin at bottom of tube using a tabletop centrifuge, and aspirate and discard supernatant.
SECTION: Bead-binding and confocal
6. Wash resin 3 times with 1 mL of room temperature buffer
SECTION: Bead-binding and confocal
7. Resuspend resin in 200 uL of buffer, then transfer to an 8-well confocal imaging chamber.
SECTION: Bead-binding and confocal
8. Focus sample and image resin. Adjust imaging parameters if necessary.
SECTION: Analysis
9. In order to measure the brightness of an individual bead, export the images to FIJI, and define an ROI that contains a bead edge. Measure this ROI, and record the maximum brightness. This brightness should correspond to the edge of the bead. Repeat this for ~20 cells, and average to acquire a single biological replicate.

Repeat experiment at least twice more to acquire two more biological replicates. Perform a T-test between the comparison groups in the experiment
TITLE: Cleaning up a biohazardous spill outside of a biosafety cabinet
AUTHORS: Steven Wilhelm, Gary LeCleir, Ashley Humphrey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">BIOLOGICAL SPILL RESPONSE </span><span>method to be used in the event of a biosafety incident. </span></div></div>

[STEPS]
?. Close off the area and allow aerosols to settle.
?. Notify others including supervisor.
?. Assemble all spill cleanup materials and review procedure.
?. Don appropriate PPE: laboratory coat, safety glasses or chemical splash goggles (depending on risk of splashes), Nitrile gloves.
?. Cover spill with paper towels.
?. Pour (don’t spray) disinfectant to contaminated surface that is covered with paper towels by pouring it around the periphery of the spill area moving inward.  Allow the appropriate contact time for the disinfectant and agent.
?. Wipe up spill. Dispose of the waste in the biohazardous waste containers.
?. Re-apply disinfectant to contaminated surface and allow it to stand for proper contact time.
?. Wipe up disinfectant, repeat if necessary.
?. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.
TITLE: Purification of anti-anti-SpA antibody (Ab-2) using SpA-bearing Staphylococcus aureus cells.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">A novel method was designed for the purification of Ab-2 from undiluted WSF of hyperimmune eggs. A total of six preparations were made. </span></div></div>

[STEPS]
?. Mix in an Eppendorf microtube 0.9 ml of undiluted WSF from immunized birds with 25 μl of SpA-bearing Staphylococcus aureus cells (Sigma-Aldrich).
?. Incubate the microtube at 37oC for 30 min.
?. After the incubation period, centrifuge the microtube in an Eppendorf 5424 centrifuge for 5 min.
?. Observe the microtube that presents a pellet of Ab-1 bound-cells at the bottom ,and the supernatant, where Ab-2 is present.
?. Decant the Ab-2 containing supernatant to another tube.
?. .  Store it at -20oC until further use.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol can be used for mapping transcriptional start site(s) (TSS) of a specific gene of interest in bacteria.</p>
<p>By slightly modifying this protocol, whole transcriptome TSS could also potentially be mapped.</p>

[BEFORE_START]
<p>Oligos used:</p>
<table style="width: 654.375px;">
<tbody>
<tr>
<td style="width: 119px;">5RACE_RNA</td>
<td style="width: 521.375px;">rGrUrG rArUrC rCrArA rCrCrG rArCrG rCrGrA rCrArA rGrCrU rArArU rGrCrA rArGrA rNrNrN</td>
</tr>
<tr>
<td style="width: 119px;">Adapter_Prim_1</td>
<td style="width: 521.375px;">TGATCCAACCGACGCGAC</td>
</tr>
<tr>
<td style="width: 119px;">Adapter_Prim_2 (nested)</td>
<td style="width: 521.375px;">ACCGACGCGACAAGCTAATGC</td>
</tr>
</tbody>
</table>
<p> </p>
<p>The image below shows three gene specific primers that anneal in the G.O.I for nested PCR. Blue marks the intergenic region to be mapped, most likely containing the TSS.</p>
<p>Gene specific primers should be carefully designed using online guidelines (e.g. <a href="https://www.thermofisher.com/de/de/home/references/protocols/nucleic-acid-amplification-and-expression-profiling/cdna-protocol/5-race-system-for-rapid-amplification-of-cdna-ends.html" target="_blank">here</a>)</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/naher2w.png" width="714" height="209" data-src="https://s3.amazonaws.com/pr-journal/nager2w.png" data-ofn="Screen Shot 2017-08-31 at 15.22.30.png" /></p>
<p> </p>
<p> </p>

[GUIDELINES]
<p>When handling RNA, samples should always be kept cold (on ice) and in an RNase-free environment. Wear gloves, wipe down surfaces with RNase-Away, use separate, RNase-free water for reactions. As a precautionary measure, samples stored long-term (&gt; 1 month) should be kept at -80 °C.</p>
<p>Before starting, check the integrity of the total RNA on a formaldehyde agarose gel to check for degradation.</p>

[STEPS]
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TITLE: Recombinant protein expression and purification of fuGFP
AUTHORS: Javiera  A Avilés, Tamara Matute, Isaac Núñez, Maira Rivera, Javiera  Reyes, Anibal Arce Medina, Cesar A Ramirez-Sarmiento, Fernan Federici

[DESCRIPTION]
This protocol has been optimized for the recombinant expression of fuGFP encoded in an open pTi vector. The plasmid encoding fuGFP used here can be found on reclone.org. The purified protein can be used for teaching about the properties of fluorescent proteins. 



[STEPS]
SECTION: DAY 1 – Plasmid transformation
1. Transform 100 ngof the open pTi plasmid containing fuGFP into E. coli BL21 (DE3) competent cells using either heat shock or electroporation.
SECTION: DAY 1 – Plasmid transformation
2. Spread transformed cells in LB Agar plates supplemented with 0.05 mg/mLKan. Grow plate overnight at 37 °C.
SECTION: DAY 2 – Preinoculum
3. Select a single colony from the LB agar plate to prepare a preinoculum in 10 mL LB media supplemented with 0.05 mg/mLKan. Grow overnight at   .

SECTION: DAY 3 – Protein Overexpression
4. Use the full volume of the preinoculum to inoculate 1 L of LB media supplemented with0.05 mg/mLKan (1% inoculation). Grow at   until reaching an optical density at 600 nm (OD600) = 0.8.
SECTION: DAY 3 – Protein Overexpression
5. Upon reaching OD600 = 0.8,  add IPTG to a final concentration of 0.5 millimolar (mM) and incubate overnight at  

SECTION: DAY 4 – Protein Purification by IMAC
6. Centrifuge the cell culture 4000 x g, 20 min, 4 °C.Then, resuspend the cell pellet in 40 mL of Buffer A freshly supplemented with 0.5 millimolar (mM) PMSF and 0.2 mg/mL lysozyme.

SECTION: DAY 4 – Protein Purification by IMAC
8. Sonicate on ice for 8 min using cycles of 1 s ON and 1 s OFF at 40% amplitude (Qsonica Q125, 125W).
SECTION: DAY 4 – Protein Purification by IMAC
9. Centrifuge the unclarified lysate 20000 x g, 20 min, 4 °C and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
SECTION: DAY 4 – Protein Purification by IMAC
10. On a 1 mL HisTrap column (GE Healthcare) pre-equilibrated with 10 column volumes (c.v.) (here, 10 mL) of Buffer A, load the supernant. Wash with 20 c.v. of Buffer B. Then, elute with 5 c.v. of Buffer C, collecting the eluted fractions every 1 mLin 1.5 ml tubes.
SECTION: DAY 4 – Protein Purification by IMAC
11. To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with 40 µLof 5X Bradford reagent and 160 µLof distilled water. Then, add 10 µLof each protein fraction and compare against a blank reference sample corresponding to 10 µLof Buffer C. You can determine your protein-containing fractions either by absorbance at 595 nm on a plate reader or visually by comparing the blue coloration of each fraction against the blank reference. Pool your fractions and collect a 10 µL sample for SDS-PAGE.
SECTION: DAY 4 – Protein Purification by IMAC
7. Incubate the resuspended cells   .
SECTION: IMAC SDS-PAGE Result
13.    

SECTION: DAY 4 – Protein Purification by IMAC
12. For storage, we suggest to do a dialysis against Buffer A, and store at 4º C.
TITLE: Quant-IT DNA Quantification (Assay)
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
Perform dsDNA quantification easily and quickly with Quant-iT dsDNA assay kits. Both the Quant-iT High-Sensitivity dsDNA Assay Kit and the Quant-iT Broad-Range dsDNA Assay Kit provide concentrated assay reagent, dilution buffer, and pre-diluted DNA standards. These DNA assay kits are highly selective for double-stranded DNA over RNA, and, in the ranges of 0.2–100 ng DNA (for the HS dsDNA Assay Kit) and 2–1000 ng DNA (for the BR dsDNA Assay Kit), the fluorescence signal is linear.

[GUIDELINES]
The Quant-iT dsDNA High-Sensitivity Assay Kit and the Quant-iT dsDNA Broad-Range Assay Kit make DNA quantification easy and accurate. The kits provide concentrated assay reagent, dilution buffer, and prediluted DNA standards. Simply dilute the reagent 1:200, load 200 µL into the wells of a microplate, add 1–20 µL sample, mix, then read the fluorescence.

The assays are highly selective for double-stranded DNA over RNA, and, in the range of 0.2–100 ng for the HS assay or 2–1000 ng for the BR assay, the fluorescence signal is linear with DNA. The assays are performed at room temperature, and the signal is stable for three hours. Common contaminants, such as salts, solvents, detergents, and protein are well tolerated in these assays.

[STEPS]
SECTION: Making Working Solution
1. Make working solution (number of samples + 24 standards + 10% = total volume).
SECTION: Making Working Solution
1.1. 200uL Buffer (per sample) + 1uL Reagent (per sample)

 

SECTION: Making Working Solution
1.2. Cover conical with a foil seal to prevent light exposure.
SECTION: Making Working Solution
1.3. Vortex working solution.
SECTION: Loading Working Solution
2. Using a 200uL 12-channel pipette and boat / reservoirs, load 200 µL of the working solution into each sample and standard well on the microplate reader plate.

SECTION: Loading Working Solution
2.1. Cover working solution with a foil seal in between uses.
SECTION: Loading Samples
3. Centrifuge the sample plate(s).
SECTION: Loading Standards
5. Add 10uL of each gamma DNA standard to each well on the microplate reader plate.

 

SECTION: Loading Samples
4. Add 1-20 µL of DNA from the sample plate(s) to each well on the microplate reader plate.

 

SECTION: Loading Standards
6. Mix sample and standard wells using a 200uL 12-channel pipette by pipetting up and down gently.

OR: Use a plate microplate vortexer on a gentle setting such as  .

SECTION: Measuring Fluorescence
7. Cover plate(s) with a foil seal and carefully carry them to a microplate reader.
SECTION: Measuring Fluorescence
8. Measure fluorescence (FL 485/528nm) using a microplate reader.
SECTION: Measuring Fluorescence
8.2. Export fluorescence table into an Excel document for future reference.
SECTION: Measuring Fluorescence
9. Use a standard curve to determine the concentration of DNA in ng/uL.

 

SECTION: Measuring Fluorescence
8.1. Set microplate type as: 96 Well Greiner Plate or other equivalent.
TITLE: Shipping wastewater samples to FDA-CFSAN
AUTHORS: Maria   Balkey

[DESCRIPTION]
This SOP provides guidance for shipping wastewater samples to the US FDA Center for Food Safety and Applied Nutrition (CFSAN), covering specific steps for registering samples with CFSAN and for the preparation of shipments (documentation, packaging, notifications). We recommend that samples are shipped in two batches, one batch after 3 months of sampling and one batch at the conclusion of your sampling (~6 months).

[BEFORE_START]
This protocol has three sections:


Section 1:  Submission of metadata 
Section 2:  Preparation of Shipping supplies
Section 3:  Sample shipment


Associated protocols:
https://www.protocols.io/view/ncbi-submission-protocol-for-sars-cov-2-wastewater-ewov14w27vr2/v5 for populating sample metadata. 


[STEPS]
SECTION: Submission of metadata
1. Fill out the BioSample custom wastewater template (extension of NCBI's Generic SARS-CoV-2: wastewater surveillance, v1.0   ) according to the NCBI submission protocol for SARS-Cov-2 protocol, once completed, send it to covidtrakr@fda.hhs.gov for registration.  Successful registrations will receive a notification confirming the sample registration, it will will include a list of CFSAN accessions.   

SECTION: Preparation of shipping supplies
2. Contact your Biosafety Officer at your institution and request instructions on how to ship wastewater samples. 
SECTION: Preparation of shipping supplies
3. Make sure adequate shipping materials are available for the preparation of your package. Prepare your shipment according to your current SOPs, check your institution and courier guidelines for the shipping of dangerous goods (wastewater samples with SARS-Cov-2) before attempting any shipment.
SECTION: Preparation of shipping supplies
4. Prepare the following documents for your package along your permits, government and/or institution requirements.
Spreadsheet with the list of samples and the description of shipment contents.
Any notes or comments regarding your package. 
SECTION: Preparation of shipping supplies
5. Label your samples accurately (tubes and caps).  Each sample (200 mL minimum) must be labeled with a CFSAN accession provided after completion of the metadata registration.  If necessary, use printed labels and secure them with transparent tape. 
SECTION: Preparation of shipping supplies
6. Wastewater samples shall be shipped in dry ice.
SECTION: Preparation of shipping supplies
7. The outer packing must be labeled accordingly. The full name and address of the sender and recipient must be included.
SECTION: Sample shipment
8. Once all the above requirements are completed, ship by express/next-day air.  Ship only on Tuesday, Wednesday or Thursday.  Ship the isolates to the address included below.


U.S. FDA CFSAN
Attn: Christopher Grim/Maria Hoffmann
5001 Campus Drive
RM 4E009
College Park, MD 20740
Christopher.Grim@fda.hhs.gov
Maria.Hoffmann@fda.hhs.gov
Phone:240-402-1080 (Maria) or 240-402-3582 (Chris)


SECTION: Sample shipment
9. Email tracking number to covidTrakr@fda.hhs.gov. Include in the notification the number of samples shipped and the corresponding list of sample identifiers.
TITLE: Staining of Gfap, Iba1,and NeuN on PFA-fixed mouse brain sections
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
Staining protocol for Gfap, Iba1, and NeuN on PFA-fixed mouse brain sections.

[GUIDELINES]
Read the full protocol before starting the procedure.

Note that this protocol uses  3 hours (room temperature) incubation for primary antibodies. 

Works with the listed antibodies and dilutions. For other references, preliminary tests are highly recommended.   


[STEPS]
SECTION: Tissue preparation and blocking
1. Take out sections from -80 and place them in an incubator/plate for 20 min  at 37 °C. This step is performed to ensure tissue attachment to the crystal slides. 

SECTION: Tissue preparation and blocking
2. 2. Draw a hydrophobic barrier on each slide using ImmEdge®Hydrophobic Barrier PAP Pen and let dry for about 10 min minutes. This will prevent buffers from escaping the tissue area.
 


SECTION: Tissue preparation and blocking
3. To initially rehydrate and permeabilize the tissue, place the slides in a slide jar containing the permeabilization solution with shaking for 30 min
 
.

SECTION: Tissue preparation and blocking
4. To prevent unspecific binding, incubate brain sections in permeabilization solution containing 5 % (v/v)   and 1 Mass / % volume  for 60 min at Room temperature.
 


SECTION: Antibody incubation
5. When blocking is finished, decant the buffer (no washing is required) and incubate primary antibodies in antibody buffer for 180 min at Room temperature according to table 1.


 

 

SECTION: Antibody incubation
6. When primary antibody incubation is finished, wash the sections 5 min with 0.05 % (v/v)    in PBS.
SECTION: Antibody incubation
7. Incubate secondary antibodies in 0.1 % (v/v)      for 60 min at Room temperature according to table 2.
 
 

SECTION: Antibody incubation
8. When secondary antibody incubation is finished, wash the sections 5 min with 0.05 % (v/v)    in PBS. Follow this with 5 min washes with PBS to remove all detergent traces.  

SECTION: Antibody incubation
9. Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop flo .
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Southeast (SE) Asia is one of the most biodiverse regions in the world and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia’s genetic diversity is still poorly characterized. The growing interest in using environmental DNA (eDNA) to assess and monitor SE Asian species, in particular, threatened mammals - has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples.</p>

[STEPS]
TITLE: Bacterial isolation and genomic DNA extraction of Mycobacterium avium from pig lymph nodes
AUTHORS: Tetsuya Komatsu, Kenji Ohya, Justice  Opare Odoi, Shota Suganuma, Kotaro Sawai, Takayuki Wada, Tomotada Iwamoto, Fumito Maruyama

[DESCRIPTION]
Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in human and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. Here, we isolated  MAH from pig lymph nodes or livers and obtained their genomes.

[STEPS]