eamag/protocols-with-steps huggingface dataset:
TITLE: Copy of       ELISA for quantification of IL-30 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>IL-31 is produced mainly by Th2 cells and dendritic cells. It is a proinflammatory cytokine and a chemotactic factor that direct polymorphonuclear cells, monocytes, and T cells to inflammatory lesions. IL-31 induces chemokines production and synthesis of IL-6, IL-16, and IL-32. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-31 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-31 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-31 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-31 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-31 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-31 standard dilutions and the human IL-31 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: A Typical DNase I Reaction (M0303)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for a typical DNase I Reaction, using the M0303 RNase-free DNase I.</div></div>

[STEPS]
?. Set up the following reaction :  AB1COMPONENTS100 μl REACTION2RNA~ 10 μg RNA3DNase I Reaction Buffer (10X)10 μl (1X)4DNAse I (RNase-free)1 μl (2 units)5Nuclease-free H2Oto 100 μl
on ice
AB1COMPONENTS100 μl REACTION2RNA~ 10 μg RNA3DNase I Reaction Buffer (10X)10 μl (1X)4DNAse I (RNase-free)1 μl (2 units)5Nuclease-free H2Oto 100 μl
?. Incubate at  for .
37 °C
?. Add  of  (to a final concentration of ).
1 µl
[EDTA]
?. Heat inactivate at  for .
75 °C
TITLE: Terminal restriction fragment length polymorphism (T-RFLP)
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
Optimized protocol for T-RFLP analysis of complex bacteria and archea communities.

[STEPS]
1. Extract and purify DNA from the sample. DNA should be at about 10-20 ng/µl.
2. Amplify the sample using PCR (refer to PCR conditions and mixes.doc). Primers need to be fluorescently labeled at their 5'. Labeling should be of at least one of the primers though both can be labeled (in which case the two primers should have different label colors).
Depending on the available size standard the optimized primers for bacteria are 27F^M^I – 908R^I or 341F^I-908R^I and for archea are 109F^I-934R^I.
It is recommended to perform 3 different PCR reactions for each sample and combine them.  PCR should be performed at minimum number of cycles and minimum annealing temp. We optimized the analysis for 3 replicates of 50 µl each at 45°C for 24 cycles (bac) and 25 cycles (arc).
3. Combine the samples and concentrate to about 50µl.
4. Perform Mung bean exonuclease digestion according to the manufacturer's instructions. Refer to Egert & Friedrich 2003, Simpson et al. 1999, or Jensen and Straus 2007 for more information.
5. Immediately clean the digested PCR reaction using PCR purification kit. Elute with 50 µl.
6. Run 3 µl of each samples on agarose gel to ensure successful cleanup and no overdigestion by the exonuclease.
7. Divide the remaining volume to 3 aliquots of 15 µl and digest overnight with 20U of restriction enzymes. For BAC we used the following: TaqI, HhaI, and RsaI. For ARC we use: TaqI, Tru1I, and MboI. For new primers refer to MICA's Enzyme resolving power analysis.
8. Desalt the samples with ethanol precipitation and resuspend in 17 µl of PCR grade DDW. Refer to Ethanol precipitation of small DNA fragments.doc
9. Run the samples on an automated microcapillary sequencer.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>SOLUTIONS &amp; BUFFERS:</strong></p>
<p> </p>
<p><em>Note: Do not use sodium azide in any buffers or solutions as sodium azide inactivates</em><br /><em>the horseradish-peroxidase enzyme.</em></p>
<p><br /><strong>Phosphate Buffered Saline (PBS):</strong><br />80.0 g NaCl<br />14.4 g Na2HPO4<br />2.4 g KH2PO4<br />2.0 g KCl<br />Add ddH2O up to 10 L; pH to 7.2 with HCl</p>
<p> </p>
<p><strong>Coating Buffer:</strong><br />Can use either Sterile PBS or<br />Sterile Carbonate Buffer (per ELISA protocol)<br />8.4 g Na HCO3<br />3.56 g Na2CO3<br />Add ddH2O up to 1.0 L, pH to 9.5.</p>
<p> </p>
<p><strong>PBS-Tween:</strong><br />0.05% Tween-20 in PBS<br />(500 μl Tween-20 in 1L PBS)</p>
<p> </p>
<p><strong>Blocking Buffer (PBS-BSA):</strong><br />1% BSA in PBS<br />(10 g BSA-Fraction V in 1L PBS)</p>
<p> </p>
<p><strong>PBS-Tween-BSA:</strong><br />1% BSA in PBS-Tween<br />(10 g BSA-Fraction V in 1L PBS-Tween)</p>
<p> </p>
<p><strong>Tissue Culture (TC) Medium:</strong><br />As appropriate for cells being analyzed</p>
<p> </p>
<p><strong>AEC Solution:</strong><br />100 mg AEC (3-amino-9-ethyl-carbazole) in 10 ml DMF<br />(N,N, Dimethylformamide)<br />Solution should be prepared in a glass tube in a fume hood.</p>
<p> </p>
<p><strong>AEC Buffer:</strong><br />(0.1 M Acetate): 148 ml 0.2 M acetic acid (11.55 ml glacial acetic acid per liter of<br />water) and 352 ml of 0.2 M sodium acetate (27.2 g per liter of water)<br />Bring up to 1L with water and adjust to pH 5.0 if required</p>
<p> </p>
<p><strong>Substrate Solution:</strong><br />800 μl AEC solution in 24 ml AEC buffer<br />Filter with 0.45 μm filter and add 12 μl 30% H2O2<br />Use immediately</p>

[STEPS]
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TITLE: Minimization of protein structures using refineD
AUTHORS: Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sometimes structural issues can be corrected with energy minimization. In this process, the weak interactions are optimized in order to produce a more thermodynamically stable structure. RefineD uses conformational sampling to try to alter homology models or other protein models to form more native like structures. This method can fix small/minor issues in the structure but cannot fix bad or inaccurate starting models. </div></div>

[STEPS]
?. [Uploading structure]
Navigate to refineD (http://watson.cse.eng.auburn.edu/refineD/)
?. [Uploading structure]
Upload your structure to the interface.
?. [Uploading structure]
Select “conservative” for refinement mode and provide your email. Then press submit.
The server takes 24 to 72 hours to run typically. I recommend copying the web address on the run page to make sure you have the information in case the email gets blocked.
?. [Analyzing refined structures]
When the results come back, navigate to the page and review the resulting structures.
There will be 1 to 5 structures returned each with two statistics. Both statistics indicate the relative change in the refined structure to the structure you submitted. Ideally they will be similar (within 5%), however it is okay if they are not.
?. [Analyzing refined structures]
Record the statistics and download the structures.  ABC1StructureGDT-HACA RMSD2132435465
ABC1StructureGDT-HACA RMSD2132435465
?. [Analyzing refined structures]
In YASARA, align your refined structures to your starting structure (the model you uploaded in step 2).
It helps to color each structure differently to tell the structures apart.
?. [Analyzing refined structures]
After alignment, look for how refineD changed the structure by minimization and record areas that changed and if possible describe how minimization changed the structure. Look at side chain packing and position of loops/secondary structure and how these features pack.
?. [Analyzing refined structures]
Submit one of your refined structures to Molprobity and assess the structure.
MolProbity protocol:  https://www.protocols.io/view/analysis-of-protein-structure-using-molprobity-bgmaju2e
?. [Analyzing refined structures]
Compare the results before and after refinement to see if some of the problems identified by Molprobity were fixed by energy minimization.
?. [Analyzing refined structures]
Name your downloaded structures using the following format:refineD_[#]_[Group_name]_[sequence_name].pdbReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence. Replace the [#] with the refineD structure number.
?. [Analyzing refined structures]
Indicate your OSF file location as a link within a note on this step.
TITLE: Amplicon clean-up using SPRI beads for RAPID nanopore kit RBK004
AUTHORS: Muhammad Faisal, Olin Silander, Nikki Freed

[STEPS]
?. Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
?. [Ampure XP bead clean up]
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add   room temperature SPRI beads to a   reaction.
50 µl
50 µl
?. [Ampure XP bead clean up]
Pulse centrifuge to collect all liquid at the bottom of the tube.
?. [Ampure XP bead clean up]
Incubate for   at room temperature.
?. [Ampure XP bead clean up]
Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. [Ampure XP bead clean up]
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. [Ampure XP bead clean up]
Add  of freshly prepared room-temperature  ethanol to the pellet.
200 µl
?. [Ampure XP bead clean up]
Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.
?. [Ampure XP bead clean up]
and repeat ethanol wash.
?. [Ampure XP bead clean up]
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
?. [Ampure XP bead clean up]
With the tube lid open incubate for  or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
?. [Ampure XP bead clean up]
Remove the tube from the magnetic rack. Resuspend pellet in  10 mM Tris-HCl pH 8.0 with 50 mM NaCl, mix gently by flicking and incubate at room temperature for .
10 µl
?. [Ampure XP bead clean up]
Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
TITLE: FAA MEDIA (FASTIDIOUS ANAEROBES AGAR)
AUTHORS: Roey Angel, Ana Lara-Rodriguez, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For the growing and maintance of Clostridium sp. </div><div class = "text-block">Fastidious Anaerobe Agar is used for the cultivation of anaerobic microorganisms and is not intended for use in the diagnosis of disease or other conditions in humans. A primary isolation medium capable of growing most clinically significant anaerobes. Developed by Lab M (Neogen® Corporation), comparisons have shown this medium to be superior to other formulations as a primary isolation medium for fastidious organisms. The peptones included have been chosen for maximum growth stimulation. Starch and sodium bicarbonate act as de-toxification agents while hemin encourages pigment production in Porphyromonas melaninogenicus. Specific growth promoting agents are Cysteine for Fusobacterium necrophorum, Propionibacterium acne and Bacteriodes fragilis, arginine for Eubacterium spp. soluble pyrophosphate for Porph. gingivalis and Porph. asaccharolyticus. Pyruvate helps neutralize hydrogen peroxide and is also utilized by Veillionella spp. as an energy source. Vitamin K and sodium succinate provide essential growth factors for some anaerobes as does the 0.1% glucose. The low level of glucose prevents the production of high levels of acids and alcohols which would inhibit colonial development.</div></div>

[STEPS]
?. Prepare stock solutions:L-cystein hydrochloride solution (50 g/l): Dissolve of Cysteine HCl Monohydrate in a   distilled water and filter sterilize. Store refrigerated. HEMIN solution (10 g/l) : Dissolve   in  ; make up to  with distilled water and filter sterilize. Store refrigerated. VITAMIN K1 solution (10 g/l): Dissolve of vitamin K1 in  and filter sterilize. Store refrigerated in a brown bottle.
0.5 g
10 mL
[Hemin]
100 µl
[NaOH]
10 mL
0.1 g
10 mL
[Ethanol]
?. For the preparation of 1L of media (50 to 66 petri dishes depending of depth) dilute in 1000 ml of distilled water:Final pH: 7.2 ± 0.2 at 25°C
[Peptone]
[Sodium Chloride]
[Soluble Starch]
[Sodium Bicarbonate]
[Glucose]
[Sodium Pyruvate]
[Sodium Pyrophosphate]
[L-Arginine]
[Sodium Succinate]
[Agar]
?. Heat to boiling to dissolve the medium completely.
?. Sterilize by autoclaving at 15 lbs pressure  for  minutes.
121 °C
?. Cool down to - and aseptically add the Cysteine HCl Monohydrate, Hemin and the Vitamin K .
50 °C
55 °C
[Hemin stock solution]
[L-Cysteine HClxH20 stock solution]
[Vitamin K1 stock solution]
?. Refrigerate the sterile medium until use (no more than 2 weeks).
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>MATERIALS:</strong></p>
<p> </p>
<p>         1)      <em>E. coli</em>, strain ____, containing plasmid</p>
<p>         2)      LB media</p>
<p>                           1.0% Bacto-tryptone</p>
<p>                           0.5% Bacto-yeast extract</p>
<p>                           1.0% NaCl</p>
<p>                           Add the appropriate antibiotic to the growth media for the plasmid to be purified.</p>
<p>         3)      Chloramphenicol, 34 mg/mL, made up in 100% EtOH, filter sterilized.</p>
<p>         4)      Spectinomycin, 10 mg/mL, made up in d-H<sub>2</sub>O, filter sterilized.</p>
<p>         5)      Lysozyme solution</p>
<p>                           25 mM Tris-HCl, pH 8.0</p>
<p>                           10 mM EDTA</p>
<p>                           50 mM Glucose</p>
<p>                           Add lysozyme to a concentration of 2 mg/mL just before use.</p>
<p>         6)      Alkaline SDS solution</p>
<p>                           0.2 M NaOH</p>
<p>                           1.0% SDS</p>
<p>                           Make up fresh from stock solutions for each use.</p>
<p>         7)      High salt solution, per 100 mL</p>
<p>                           60.0 mL of 5 M KOAc</p>
<p>                           11.5 mL of glacial acetic acid</p>
<p>                           28.5 mL of d-H<sub>2</sub>O</p>
<p>         8)      50 mM Tris-HCl, pH 8.0, 100 mM NaOAc</p>
<p>         9)      10 mM Tris-HCl, pH 8.0, 1 mM EDTA (1X TE buffer)</p>
<p>         10)    Ethidium bromide, 10 mg/mL in d-H<sub>2</sub>O</p>
<p>         11)    CsCl, solid</p>
<p>         12)    100% EtOH</p>
<p>         13)    Isopropanol, CsCl/TE buffer saturated</p>
<p>         14)    3 M NaOAc</p>
<p>         15)    Beckman Ti50 rotor quick seal tubes</p>
<p>         16)    Syringes, with 18g needles</p>
<p>         17)    Phenol, buffer-saturated<br /><br /><strong>References</strong><br /><br /></p>
<ul>
<li>C. Birnboim. A rapid Alkaline Extraction Method for the Isolation of Plasmid DNA.</li>
</ul>
<p>         <u>Methods In Enzymology</u> <strong>100</strong>:  243-255.</p>
<p> </p>
<ul>
<li>Maniatis, E.F. Fritsch, and J. Sambrook. Large-scale Isolation of Plasmid DNA.</li>
</ul>
<p>         <u>Molecular Cloning:  A Laboratory Manual.</u>  Pages 86- 94.</p>

[STEPS]
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TITLE: Midbrain astrocyte and co-culture
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Midbrain astrocyte and co-culture for mix-genetic experiments.

[STEPS]
SECTION: Intro
1. Midbrain NPC were generated following the publish protocol https://www.protocols.io/view/midbrain-organoid-differentiation-in-spinner-flask-rm7vzbnr4vx1/v1 with modifications listed bellow.
SECTION: NPC generation
2. From a 80% confluent plate of iPSCs in a 10 cm plate.
SECTION: NPC generation
3. Wash with 5ml of PBS
SECTION: NPC generation
4. For the passaging add 3 mL of   

SECTION: NPC generation
5. Add 5 mL  of StemFlex , gently mix and transfer to a 15 ml conical tube.
SECTION: NPC generation
6. 300 rcf, 3 min, 25 °C 

SECTION: NPC generation
7. Resuspend the cells in 1 mL of StemFlex with rock inhibitor and Count the cells using 
 

SECTION: NPC generation
8. Plate 2 million cells in a 1/100    coated plate in Stemflex with 10 μM Y-27632.

SECTION: NPC generation
9. D0 change half the media with D0-1 medium.
SECTION: NPC generation
10. D2 change half the media with D2-3 medium.
SECTION: NPC generation
11. D3 Change half the media with D2-3 medium.
SECTION: NPC generation
12. D4 change half the media with D4-7 medium.
SECTION: NPC generation
13. D5 change half the media with D4-7 medium.
SECTION: NPC generation
14. D6 change half the media with D4-7 medium.
SECTION: NPC generation
15. D7 change half the media with D4-7 medium.
SECTION: NPC generation
16. D8 change all the media with D8-11 medium.
SECTION: NPC generation
17. D9 change half the media with D8-11 medium.
SECTION: NPC generation
18. D10 change half the media with D8-11 medium.
SECTION: NPC generation
19. D11 change half the media with D8-11 medium.
SECTION: NPC generation
20. Freeze the NPCs in   

SECTION: Astrocyte co-culture
25. In a 96 well plate coated with   1/100 plate 50k NPCs

SECTION: NPC Plating
21. Thaw the NPCs and transfer to a 15 ml conical tube.
SECTION: NPC Plating
22. Add 5 ml of D12 terminal media with 10 μM Y-27632
SECTION: NPC Plating
23. 300 rcf, 3 min, 25 °C 

SECTION: NPC Plating
24. Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using 
 

SECTION: Astrocyte co-culture
26. Change half media every day until day 25.
SECTION: Astrocyte co-culture
27. Obtain astrocytes following protocol https://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2
SECTION: Astrocyte co-culture
28. Thaw the Astrocytes and transfer to a 15 ml conical tube.
SECTION: Astrocyte co-culture
29. Add 5 ml of A   with 10 μM Y-27632.

SECTION: Astrocyte co-culture
30. 300 rcf, 3 min, 25 °C 

SECTION: Astrocyte co-culture
31. Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using 
 

SECTION: Astrocyte co-culture
32. Plate 10k on the neurons.

SECTION: Astrocyte co-culture
33. Mature the co-cultures until day 50 changing media every other day with day 25 media.
TITLE: Outcomes of Total Hip Replacement in Septic Arthritis of the Native Hip Joint Amongst the Adult Population: A Systematic Review
AUTHORS: Teddy Cheong, Surya Varma Selvakumar, Ryan Kwang Jin Goh, Ing How Moo

[DESCRIPTION]
Background: Septic arthritis is a debilitating condition that results in joint destruction and irreversible loss of joint function. Surgical treatment options include arthroscopy, resection arthroplasty and total hip replacement (THR). There is existing literature on the treatment of septic arthritis of various joints including periprosthetic or native joints. However, to our knowledge, there is a lack of a consolidated review that focuses solely on THR outcomes in a previously infected native hip. This study aims to be the first systematic review that examines clinical outcomes of THR, specifically in septic arthritis of the native hip joint in the adult population.

Methodology: This systematic review will be conducted in accordance with relevant requirements of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement. PubMed, Embase, Cumulative Index to Nursing and Allied Health Literature (CINAHL), SCOPUS, Cochrane Library, grey literature, and bibliographic references will be searched from inception to October 2023. Only case series or cohort studies published within the last 20 years assessing the outcomes of total hip replacement in native hip septic arthritis will be included. Literature retrieval and data extraction will be conducted by three independent reviewers. The overall methodological quality of the included studies will be assessed using the methodological index for non-randomized studies (MINORS) tool. Re-infection rate and various functional outcomes data such as the Harris Hip Score (HHS), Visual Analogue Scale (VAS), Merle D'Aubigne and Postel (MAP), Western Ontario, McMaster Universities Arthritis Index (WOMAC) Short Form 12-Item (SF-12) scores will be collected and analysed. 

Expected Results: Expected to have 5-10 studies included after removal of duplicates and implementation of inclusion and exclusion criteria. Compiled results of re-infection rates and outcomes of THR in native septic arthritis of the hip will be reported and analysed. 


[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the transformation of Synechococcus sp. PCC7002 with linear DNA fragments with homologous regions. </p>

[GUIDELINES]
<p>Work as sterile as possible.</p>

[STEPS]
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TITLE: Retro-orbital injection of virus or dye in mice
AUTHORS: Boaz Mohar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is following:</div></div>

[STEPS]
?. [Injection]
Prepare either virus dilution (5.0e11-1.0e12) with PBS prepare or a dye aliquot. Injection is up to 200ul per mouse.
?. [Injection]
Take one mouse from the cage into an induction chamber and start isoflurane at 3% with flow rate of ~1L/min
?. [Injection]
After the mouse has slow breathing (~1/s) move the mouse to a nose cone delivery of isoflurane at 1.5% with around the same flow rate.
?. [Injection]
Expose the eye ball with two fingers (above and below) and make sure the you are not pressing on the trachea and the mouse's breathing is regular.
?. [Injection]
Use a 29G syringe at a 30-degree angle beveled downwards until you hit the bone, retract a bit and slowly inject. See cited paper for more details.
?. [Injection]
Remove the needle slowly and press the eyelid shut for a few seconds.
?. [Injection]
Validating the injection was successful is possible with JF525 by looking at the pee of the animal after it wakes up.Leaving the animal in a clean cup with kim-wips will collect the pee and it would seem pink.