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

[GUIDELINES]
<table width="149">
<tbody>
<tr>
<td width="83">Tris-HCl pH7.4</td>
<td width="66">50 mM</td>
</tr>
<tr>
<td>NaCl</td>
<td>150 mM</td>
</tr>
<tr>
<td>Sodium Deoxycholate</td>
<td>1 %</td>
</tr>
<tr>
<td>SDS</td>
<td>0.10 %</td>
</tr>
<tr>
<td>Triton-X100</td>
<td>1 %</td>
</tr>
<tr>
<td>EDTA</td>
<td>0.5 mM</td>
</tr>
<tr>
<td>Na3VO4</td>
<td>1 mM</td>
</tr>
<tr>
<td>NaF</td>
<td>50 mM</td>
</tr>
</tbody>
</table>
<br />Sterilize by passing&nbsp;&nbsp;through 0.22 &micro;m filter. Store at 4 &deg;C. Add protease inhibitor cocktails just before using.

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
This protocol assumes that salt extracts lysin from the virion rather than from cell walls. Yield seems to confirm this assumption.<br /><br />Quality control: lysin assay shows that all chlorophyll has been released and no cell wall material pellets at low speed (wall has been solubilized).<br /><br />The final high speed pellet (after step 8) showed a white layer (virus) remaining in the pellet.

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

[DESCRIPTION]
Version 1b<br />17 October 2012<br /><br />This protocol decribes the extraction of DNA from viral particles using Wizard Prep Resin and Columns from Promega.

[GUIDELINES]
This protocol is part of a larger collection of Cesium-Chloride related protocols. This is number (3) of (4):<br />1. <a href="https://www.protocols.io/view/Cesium-Chloride-gradients-and-DNA-Extraction-Virus-c7dzi5" target="_blank">Cesium Chloride Gradients</a><br />2. <a href="https://www.protocols.io/view/CsCl-Step-Gradient-to-Purify-Phage-c4zyx5" target="_blank">CsCl Step Gradient to Purify Phage</a><br />3. <a href="https://www.protocols.io/view/DNA-Extraction-of-Viruses-using-Wizard-Prep-Column-c26yhd" target="_blank">Cesium Chloride and DNA Extraction of Viruses using Wizard Prep Columns</a><br />4. <a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm?guidelines" target="_blank">Cesium Chloride Dialysis for Viruses<br /><br /></a>Needed:<br />
<ul>
<li>1 mL DNA Purification Resin</li>
<li>0.5 mL CsCl purified virus</li>
<li>Minicolumn</li>
<li>3ml or 5ml sterile syringe with plunger removed</li>
<li>80% isopropanol</li>
<li>1.5 mL centrifuge tube</li>
<li>Mico-centrifuge @ 10,000g</li>
<li>TE Buffer (10mM Tris-Cl, pH8.0, 1mM EDTA)</li>
<li>Quant-iT PicoGreen dsDNA assay kit</li>
</ul>
<br />To prepare a CsCl solution of a particular density, the percent by weight of CsCl can be calculated by the formula:<br />% wt/wt = 137.48 - 138.11/p<br />where 'p' is the desired density. For example, for p = 1.7 g/mL, use 56.24g CsCl and 43.76 mL H2O.

[STEPS]
?. 
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?.
TITLE: Cryopreservation of microalgal cultures
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for long-term .cryopreservation of microalgal cell cultures.</div></div>

[STEPS]
?. [Preparing the cryoprotectant]
Make a  solution of DMSO in the appropriate culture medium for the strains being preserved.Eg.  medium and   to make  cryoprotectant.
[culture medium]
[DMSO]
50 mL
Work in the fume hood when using DMSO.
?. [Preparing the cultures]
Prepare the cryotubes with a 1:1 ratio of cryoprotectant:cultureEg.  and  for a 2 mL cryotube.
[cryoprotectant]
[culture]
?. [Preparing the cryoprotectant]
Filter sterilise the cryoprotectant using a 0.2 μM filter.
?. [Preparing the cultures]
Once the cryoprotectant and culture have been mixed in the cryotubes, allow  for the cryoprotectant to penetrate.
?. [Progressive freezing]
Place the cryotubes into a Mr Frosty freezing container (filled to the line with isopropyl alcohol/isopropanol/propan-2-ol).
?. [Progressive freezing]
Place the freezing container in a  freezer  .
-80 °C
?. [Progressive freezing]
Remove the tubes from the freezer and flash freeze them in liquid nitrogen.
?. [Progressive freezing]
Store the tubes either in liquid nitrogen or in a  freezer for as long as required.
-150 °C
?. [Culture recovery]
To recover a cryopreserved culture, thaw the cryotubes in a water bath pre-heated to  to prevent ice crystals from re-forming as they thaw.
40 °C
?. [Culture recovery]
Add the defrosted culture to  .
[fresh culture medium]
?. [Culture recovery]
Place the culture at the appropriate temperature but keep it in the dark for  , for example by wrapping the culture flask in aluminium foil.
?. [Culture recovery]
After  , remove the aluminium foil. Monitor the culture to determine if recovery has been successful.
TITLE: Prospective Human Liver, Adipose and Blood Collection v.2 -- University of Minnesota TMC
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Collection protocol obtained from the attached BioNet Specimen Procurement Agreement provided by the UMN CTSI Biorepository and Laboratory Services (BLS). *Updated protocol as of 11.7.2023. 

  


[STEPS]
TITLE: SOLUTION- 02 - Phosphate Buffered Saline (PBS)
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recepe is used in the following protocols:</div><div class = "text-block">- Separation and purification of human PBMC from FRESH BLOOD</div><div class = "text-block">- Separation and purification of human PBMC from BUFFY COAT</div><div class = "text-block">- Magnetic bead-based CD4+ T cell isolation from PBMCs with Dynabeads: CD4 Positive Isolation Kit</div><div class = "text-block">- Magnetic bead-based TREG-TEFF cell isolation from PBMC with Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit</div><div class = "text-block">- Staining of human PBMC or ISOLATED SUBSETS with Cell Proliferation Dye-eFluor™ 670 (CPD-eFluor670) for cell proliferation evaluation by Flow Cytometry</div></div>

[STEPS]
TITLE: Elevated body swing test in rats
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing the elevated body swing test in rats. The elevated body swing evaluates motor asymmetry in body swings when the rat is briefly hanged by the tail, and can be used in animal models with unilateral lesions of the nigrostriatal dopaminergic pathway.

[STEPS]
SECTION: Test
1. Bring cages to the behavioral room for at least 60 min prior to the test for habituation
SECTION: Test
2. Place rat individually in a new cage
SECTION: Test
3. Wait until attains neutral position with all four limbs touching the bottom of the cage
SECTION: Test
4. Lift vertically by the base of the tail
SECTION: Test
5. Record the first direction of body deviation away from the vertical axis of 30 degree during a maximum 5 s interval

SECTION: Test
6. Repeat the test five times, with a brief rest in between
TITLE: Convergence model for effectual prevention and control of zoonotic diseases: a health system study on ‘One Health’ approach in Ahmedabad, India
AUTHORS: Sandul Yasobant, Walter Bruchhausen, Deepak Saxena, Timo Falkenberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The complexity and increasing burden of zoonotic diseases create challenges for the health systems of developing nations. Public health systems must therefore be prepared to face existing and future disease threats at the human-animal interface. The key for this is coordinated action between the human and the animal health systems. Although some studies deal with the question of how these two systems interact during unforeseen circumstances such as outbreaks, a dearth of literature exists on how these systems interact on early detection, prevention and control of zoonotic diseases; assessing this problem from the health system perspective in a developing nation adds further complexity. Systems thinking is one of the promising approaches in understanding the factors that influence the system's complexity and dynamics of health maintenance. Therefore, this study aims to understand the generic structure and complexity of interaction between these actors within the domain of One Health for the effectual prevention and control of zoonotic diseases in India.The present study will be executed in Ahmedabad, located on the Western part of India, in Gujarat state, using a mixed methods approach. For the first step, zoonotic diseases will be prioritised for the local context through semi-quantitative tools. Secondly, utilising semi-structured interviews, stakeholders from the human and animal health systems will be identified and ranked. Thirdly, the identified stakeholders will be questioned regarding the current strength of interactions at various levels of the health system (i.e. managerial, provider and community level) through a quantitative network survey. Fourthly, utilising a vignette method, the ideal convergence strategies will be documented and validated through policy Delphi techniques. Finally, through a participatory workshop, the factors that influence convergence for the control and prevention of zoonotic diseases will be captured.This study will provide a comprehensive picture of the current strength of collaboration and network depth at various levels of the health system. Further, it will assist different actors in identifying the relevance of possible One Health entry points for participation, i.e. it will not only contribute but will also develop a system convergence model for the effectual prevention and control of zoonotic diseases.</div></div>

[STEPS]
TITLE: Toxicity Assay
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Toxicity Assay (based on growth curves protocol)</div></div>

[STEPS]
?. [Rules of thumb]
Never remove > 1/3 of a culture for a growth curve
?. [Rules of thumb]
Read OD600 (optical density at 600 nm) of 200 microliters of culture added to 800 microliters of fresh media -> 1,000 microliters of fluid in cuvette; = 1:5 dilution
Remember to set the factor equal to 5 when reading from the spectrometer
?. [Rules of thumb]
Easier to measure 800 microliters of fresh media into cuvettes ahead of time; simply store in cuvette box
?. [Rules of thumb]
Easier to measure media into a 50 mL conical for filling fresh cuvettes; less likely to get a contaminated bottle
?. [Rules of thumb]
Easier to use same blank multiple times; suggest every time you refill conical with media for cuvettes, make a new blank with that and use with cuvettes from that mixture.
?. [Making an overnight culture]
Measure 5mL appropriate media with appropriate antibiotics into fresh conical (make sure to label conical with date, strain!)
?. [Making an overnight culture]
Take 1 colony from a plate with appropriate strain (use pipette to poke that colony)
?. [Making an overnight culture]
Inoculate media
?. [Making an overnight culture]
Grow overnight (usually start culture ~3-5 p.m. night before culture is needed)  In the morning:
?. [In the morning]
Measure OD600
?. [In the morning]
Use V1C1=V2C2 to make a 0.01 OD600 dilution in media for growth curve
?. [In the morning]
Place culture in flasks, mix, place in the appropriate incubator, start count up timer.
?. [Toxicity Assay:]
The first time point is at 30 min on the count-up timer.
?. [Toxicity Assay:]
Take time point every 30 min, write down data and graph as you go
?. [Toxicity Assay:]
Use count up timer to measure time. When it reaches 30 min, 1 hour, 1.5 hours, etc. remove cultures from the incubator, pipette into cuvettes (remember 1/5 dilution, e.g. 200 microliters culture in 800 microliters media)
?. [Toxicity Assay:]
Remember to mix culture gently before adding to a cuvette  (the culture can be clumpy at the bottom, and you want a homogenous solution)
?. [Toxicity Assay:]
Once the culture reaches OD 0.3 (at about 2-3 hours for E. coli in LB), split cell culture in half.
Try to reach as close to OD 0.3 as possible. May require you to take samples off the 30-minute cycle.
?. [Toxicity Assay:]
Induce ½ cells with the appropriate inducer (for example, arabinose) at the appropriate final concentration (for ara, 0.2% is saturating).
?. [Toxicity Assay:]
Continue incubation for all cultures, taking OD600 every 30 minutes after induction.
?. [Rules of thumb]
Place cuvettes into 10% bleach solution when finished. *This is a must for BSL 2 organisms such as E. faecalis and EHEC.
TITLE: Ward lab alkaline bleaching protocol
AUTHORS: Jordan Ward

[DESCRIPTION]
Current protocol for how Ward lab synchronizes C. elegans by alkaline bleaching.

[STEPS]
1. Add M9+0.05% gelatin to nematode plates containing gravid adults, swirl the plate, recover the liquid with the worms and transfer to centrifuge tubes. For 10 cm plates, pour enough M9+0.05% gelatin to cover the surface of the plate and transfer to a sterile 15 ml conical tube.
2. Let the worms sink to the bottom of the tube and aspirate the liquid leaving 0.5 ml. Add M9+0.05% gelatin to 10 ml, and either let settle by gravity or spin at 1000g for 1 minute. Aspirate to 2 ml.
3. Prepare bleaching solution. Per bleaching reaction:
 
10 M NaOHul200bleachul500dH2Oul1300Total2000
 
Scale accordingly for the number of bleaching reactions. We only bleach six strains at a time to avoid over-bleaching.
4. Add 2 ml bleaching solution to the 2 ml solution of worms. Either vortex or  vigorously shake for 5 minutes, checking worms every minute. Once bodies are dissolved  add M9 to 14 ml, centrifuge at 1000 g for 60 sec and aspirate liquid. Note: after the first centrifugation a film can be seen over the eggs. We usually aspirate less liquid that time, leaving 1  ml, and reduce the volume left for the following centrifugations.
5. Repeat for a total of 5 washes. For each wash, cap the tube and shake briefly before centrifuging  at 1000 g for 60 sec and aspirating the liquid to 500 µl. For the first two washes bring volume to 10 ml with M9 , for the last three washes use 5 ml of M9.
6. After the final wash, centrifuge at 1000 g for 60 sec and aspirate the liquid to 500 µl. Bring volume to 7 ml with M9+5 µg/ml cholesterol. Rotate tube overnight at 20ºC.

7. The next day count the number of worms in a 5 µl drop to determine the number of L1s per ml. If density is low, increase the volume of the drop. If density is high, decrease the volume of the drop. Centrifuge at 1000 g for 60 sec and aspirate the liquid to 500 µl. Add 1 ml of M9+0.05% gelatin. Transfer to a 1.5 ml tube. Spin 700 x g for 1 minute in a microfuge. Aspirate to 100 µl.
8. Resuspend pellet but pipetting and plate on desired number and type of plates
TITLE: What does the latest generation CAR-T look like?
AUTHORS: Alex Brown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In the past decade, new advances in tumor treatment have been made. Immunotherapy works by activating the patient's own immune system to target cancer cells. CAR-T cell therapy has shown significant activity in certain cancers, and two CAR-T therapies, Kymriah and Yescarta, have been approved by the FDA for the treatment of acute lymphoblastic leukemia and large B-cell lymphoma.</div><div class = "text-block"><span>However, despite advances in CAR-T cell therapy, scientists have not yet fully determined how CAR-T cells function in structure. Recently, researchers from the Moffitt Cancer Center in the United States have solved this problem and described the mechanism of action of these new drugs. The results were published in </span><span style = "font-style:italic;">Science Signaling</span><span>. it still remains a big challenge in the field of </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">drug discovery</span></a><span style = ":;">.</span></div></div>

[STEPS]
TITLE: Standard Interview of Evidence Use (SIEU)
AUTHORS: Drew Gitomer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> This is a modification of Palinkas' SIEU - we removed items 16-20 because they were not appropriate for an educational setting</div><div class = "text-block">We also changed language to be more consistent with educational terminology (e.g., Agency changed to School Distrct)</div></div>

[STEPS]
TITLE: Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism - ASAP protocol collection
AUTHORS: michela.deleidi, Pascale Baden, María José Pérez J., Hariam Raji, Federico   Bertoli

[DESCRIPTION]
Collection of protocols of Deleidi Lab used in the publication: "Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism"

[STEPS]
TITLE: ENNOLIFE SARS-CoV-2 Antigen Test Kit Protocol
AUTHORS: Hsiao-Chung Tsai

[STEPS]
?. [Setup ENNOLIFE Clinical Chemistry Analyzer]
Keyin Patient IDs then press “START” to check the connection of Analyzer and Notebook.
?. [Running a nasal swab sample]
For specimen collection of nasal swabs, follow the CDC Swab Collection Guidelines and swab manufacturers' recmommandations.  Use a flocked tapered swab. Tilt patient’s head back 70 degrees. While gently rotating the swab, insert swab less than one inch (about 2 cm) into nostril (until resistance is met at turbinates). Rotate the swab several times against nasal wall and repeat in other nostril using the same swab.
?. [Running a nasal swab sample]
Place and soak the Patient Swab into the Tube R1 (extraction buffer).  Rotate and stir up and down the swab for 15 secs then standing for 1 min. Mix Tube R1 for several times by swab and discard the swab in biohazard waste.
?. [Running a nasal swab sample]
Transfer solution  to the Tube R2.
[from Tube R1]
?. [Running a nasal swab sample]
Mix Tube R2 by a vortex mixer.
25 33
Make sure all beads are suspended well.
?. [Running a nasal swab sample]
Insert Tube R2 into carrier of vortex mixer and mix for 15 min.
25 33
?. [Running a nasal swab sample]
Spin down Tube R2. Insert Tube R2 into Magnetic Rack and stand for 30 secs.
Centrifuge: 5000 33, 10 s, 25 10
?. [Running a nasal swab sample]
Remove supernantant of Tube R2.
Attention, do not remove any magnetic beads.
?. [Running a nasal swab sample]
Add 1000 uL Wash Buffer to Tube R2. Mix for 10 secs by a vortex mixer.
[Wash buffer]
25 33
?. [Running a nasal swab sample]
Spin down Tube R2 for 10 secs. Insert Tube R2 into Magnetic Rack and stand for 30 secs.
Centrifuge: 5000 33, 10 s, 25 10
?. [Running a nasal swab sample]
Remove supernantant of Tube R2.
Attention, do not remove any magnetic beads.
?. [Running a nasal swab sample]
Transfer solution  to the Tube R2.Mix for 10 secs by a vortex mixer.
[from Tube R3]
25 33
?. [Running a nasal swab sample]
Transfer   (including beads) into the Reagent Disc.
[from Tube R2]
?. [Running a nasal swab sample]
On the Mainpage of the Analyzer(1) Select “Measurement”. (2) Select “Swab”. (3) Select “START”.
?. [Running a nasal swab sample]
After 10 mins, software will show the results.
?. [Setup ENNOLIFE Clinical Chemistry Analyzer]
Make sure ENNOLIFE Clinical Chemistry Analyzer and Notebook was connected according to the operation manual. Double click "ENNOLIFE COVID-19 software"  shortcut from the desktop.
?. [Running a nasal swab sample]
While waiting, open pounch and setup the reagent disc into holder.  Install the holder on the Analyzer.
TITLE: Thiobarbituric acid reactive substances (TBARS) Assay
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Plasma concentrations of thiobarbituric acid reactive substances (TBARS) are an index of lipid peroxidation and oxidative stress. The protocol describes how the DiaComp quantitates TBARS in the animal models.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bizf4kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation: ]
Plasma:• Place 100µL plasma into a labeled 1.5mL micro-centrifuge tube. Tissue:• Label 1 sets of 1.5mL micro-centrifuge tubes, 1 set screw top tubes and 1 set of 0.5mL tubes. • Weighed out ~20mg and sonicate in 200µL RIPA buffer + inhibitors. • Sonicate. • Centrifuged @ 3000 for 10 min @ 4º . 1. Remove 10 µL aliquot into the 0.5mL tubes for protein analysis. 2. Place 100 µL lysate into a labeled 1.5mL micro-centrifuge tube. 3. Add 200µL ice cold 10% Trichloroacetic acid to precipitate protein. 4. Incubate for 15 minutes on ice. 5. Prepare standards as follows: 6. Centrifuge samples @ 2200 x g for 15 min. at @ 4ºC.7. Place 200µL supernatant and standards into new labeled screw top 1.5ml tube. 8. Add and equal volume of 0.67% (w/v) TBA. 9. Incubate in a boiling water bath for 10 min. 10. Cool. Sample is ready for assay.
?. [Performing Assay:]
1. While samples are cooling, layout on computer and save as TBxxxxxx.sed where xxxxxx is the date in yyddmm format. 2. Load 150 µL into each standard well in duplicate. 3. Load 150 µL into each samples well in duplicate. 4. Put in plate reader and press start.
?. [Reading the Plate:]
• Record absorbance at 532 nm.  1. Turn on Multiskan and open your saved file TBxxxxxx.sed.  2. Place plate onto Multiskan holder and click START. 3. Select Process>Curve Fit. Choose the appropriate data (usually Measure1), then click OK. 4. Save Curve Fit data sheet as an Excel file into the Data folder/TBARS data folder. Use the naming convention TBxxxxxx.xls, where xxxxxx is the date in yymmdd format.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In the past, identifying a gene of interest for a mutant phenotype in maize has proven to be a laborious process. Now, with the aid of next generation sequencing technology, the researcher can quickly identify the region containing a mutant gene and in some cases identify the causative lesion. In our BSA-seq protocol, we use the Galaxy platform for file processing and manipulation. This platform is intended to provide a user friendly experience, so that researchers of any level can clone maize genes. We start with raw sequencing reads from our F2 mapping population and end with plots of SNPs on each chromosome and a list of candidate SNPs. The Galaxy platform generates the files needed for plotting SNPs with the R package ggplot2 and for identifying candidate SNPs with SnpEff. Our protocol will guide the user step-by-step through the Galaxy platform, chromosome plotting, and SnpEff. </p>

[STEPS]
?. 
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TITLE: CTAB DNA Extraction for genotyping
AUTHORS: Yaowu Yuan

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

[STEPS]
?. Grind fresh plant tissue with liquid nitrogen or silica-gel dried tissue in a 1.5 ml Eppie tube.
A little silica gel grains in the tube actually helps the grinding.
?. Add .
[CTAB DNA Extraction buffer]
?. Incubate the CTAB/plant extract mixture for  at  in the heat block and invert to mix throughout the 15 minutes.
?. Add : IsoAmyl Alcohol (24:1) in the hood and mix the solution by invertingthe tubes (do not vortex).
[Chloroform]
?. Centrifuge at  for .
?. Transfer the upper aqueous phase only to a new eppie tube (~).
500 µl
?. Add  followed by  and invert to mix.
[7.5M Ammonium acetate]
[ice cold 100% ethanol]
?. Put tubes in  freezer for  (or longer) to precipitate the DNA.
?. Centrifuge at   for .
You should see a pellet at the bottom (align the tubesso that you know where the pellet is in case you can’t see it very well).
?. Remove the supernatant and wash the DNA pellet as follows. (1/2)
?. Add  . (1/2)
[ice cold 70% ethanol]
?. Centrifuge at   for . (1/2)
?. Remove the supernatant and wash the DNA pellet as follows. (2/2)
?. Add  . (2/2)
[ice cold 70% ethanol]
?. Centrifuge at   for . (2/2)
?. Remove all the supernatant and allow the DNA pellet to dry in the hood (approx. ).
Do not over dry the pellet since it will be hard to re-dissolve.
?. Resuspend the DNA in .
[dH2O]
?. Run the DNA on a 1.0% agarose gel to check the quality of the DNA.