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

[DESCRIPTION]
<p>Polyol dehydrogenases are enzymes that convert polyalcohols into sugars, using NAD<sup>+</sup> as hydrogen acceptor. Sorbitol, mannitol or other polyalcohols can be used as substrates and NADH is produced. In this assay, proteins are first separated by native polyacrylamide gel electrophoresis and the gel is then incubated with the enzyme substrates. NADH produced by the dehydrogenase reduces tetranitro blue tetrazolium chloride (TNBT), generating a black product that stains the gel. This procedure gave good results with polyol dehydrogenases from the digestive gland of gastropods and, with the proper adaptations, can be applied to other dehydrogenases and other organisms.</p>

[BEFORE_START]
<p>Prepare the following solutions:</p>
<p> </p>
<p><strong>Homogenization medium for marine gastropods</strong></p>
<p>(adapted from Stewart et al. 1994 and Moyers et al. 1991)</p>
<p>Sucrose - 500 mM</p>
<p>Potassium chloride - 150 mM</p>
<p>Ethylenediamine tetraacetic acid (EDTA), sodium salt - 1 mM</p>
<p>Tris-HCl 50 mM pH 7.4</p>
<p> </p>
<p>This medium, without phenylmethylsulfonyl fluoride (PMSF), can be stored at -20º C in aliquots. To avoid the inactivation of PMSF in <em>aqueous solution,</em> add 5 µl of 200 mM PMSF (in absolute ethanol) per ml of medium just before use (final PMSF concentration 1 mM).</p>
<p> </p>
<p><strong>Incubation Medium</strong></p>
<p>(adapted from Lewis and Gibson, 1978).</p>
<p>Phenazine methosulphate - 0.01 mg . ml<sup>-1</sup></p>
<p>Tetranitro blue tetrazolium chloride (TNBT) - 0.1 mg . ml<sup>-1</sup></p>
<p>NAD<sup>+ </sup>- 3 mg . ml<sup>-1</sup> (» 4.5 mM)</p>
<p>Polyol substrate (mannitol, sorbitol or other polyol) - 50 mM</p>
<p>50 mM Tris-HCl, pH 8.0 (TNBT precipitates at higher pH)</p>
<p> </p>
<p><strong>Sample buffer for native gel electrophoresis</strong></p>
<p>125 mM Tris-HCl, pH 6.8</p>
<p>80% glycerol </p>
<p>0.02% Bromophenol Blue</p>

[STEPS]
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TITLE: Euplotes crassus transfection using FuGene HD Transfection Reagent as vehicle (provisional)
AUTHORS: RACHELE CESARONI, Rachele Cesaroni

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

[STEPS]
?. Collect 4 x 104 well-fed Euplotes crassus cells (we used E.coli as the only food source) by centrifugation at 400 rcf for 3 minutes.
?. Wash the cells twice with artificial sea water (see attachment for the recipe) and once with 500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0 (400 rcf for 3 minutes each time). Then resuspend Euplotes crassus cells in 50 μl of the medium (500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0). To get this small volume you may require an additional minute of centrifugation.
Recipe for complete sea water (1 L):

36 g Reef Crystals
1 ml Walne's solution
1 ml of 10 μg/ml FeSO40.2 ml of 2 mg/ml thiamine (light sensitive; store at 4 ºC)
Add distilled water up to 1 L
?. Transfer the cells into wells within a 96-well plate for transfection.
?. Allow FuGene HD Transfection Reagent to reach room temperature, and then mix it by inverting or vortexing briefly (if you see a precipitate, briefly warm it at 37°C, and then let it reach room temperature).
?. Add 90-98 μl of medium (500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0) at room temperature and 4 μg of DNA in MilliQ H2O (0.2-1 μg/μL) to an Eppendorf tube and vortex (after adding the DNA the final volume must be 100 μl).
?. Add 6 μl of FuGene HD Transfection Reagent directly to the medium and mix immediately.
?. Icubate FuGene HD Transfection Reagent and DNA mixture for 15 minutes at room temperature.
?. Add 10 μl of the FuGene HD Transfection Reagent and DNA mixture to the 50 μl of Euplotes crassus cells in medium (mix everything by pipetting).
?. One hour after addition of FuGene/DNA complexes, add 50 μl of artificial sea water to the cells.
?. After another hour, add to the cells other 50 μl of artificial sea water.
?. An hour later harvest the cells (400 rcf for 3 minutes), and wash them twice with artificial sea water (400 rcf for 3 min each time). Then resuspend them in 400 μl of the artificial sea water.
?. Transfer the cells into glass depression wells for subsequent monitoring.
?. Incubate the cells at 24°C, then examine them by fluorescence microscopy to determine expression of the construct.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was used to determine when to collect sex biased samples for RNA extraction and establish expression profile throughout mealybug development.</p>
<p> </p>
<p>Sex ratio throughout oviposition was established first in Planococcus citri (Ross et al., 2010). We used the procotol that the authors established and tried it on Planococcus kraunhiae. The sex ratio variation follow a similar pattern (Vea et al., 2016).</p>

[STEPS]
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TITLE: Immunoprecipitation assay
AUTHORS: Ning Zhang , Peng Gao

[STEPS]
?. To harvest cells in nondenaturing conditions, remove media and rinse cells with ice cold 1×PBS
?. Remove PBS and add 0.5 ml ice cold cell lysis buffer to each plate(10cm) and incubate on ice for 5 min
?. native protein: Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. Sonicate on ice 3 times for 5 sec each. [denatured condition: scrape cells off the plate and transfer to 2.5 ml tube, boil for 10 min, then add 4 volumes cell lysis buffer, this is the cell lysate. ]
?. Native protein : Microcentrifuge for 10 min at 4 ℃,14000g and transfer the supernatant to a new tube. The supernatant is the cell lysates, store at -80℃.
?. add primary antibody(at the appropriate dilution as recommended in the product datasheet) to 200ul cell lysate at 1mg/ml. incubate with gentle rocking overnight at 4℃.
?. Add protein A/G agarose beads (10-30ul of 50% bead slurry). Incubate with gentle rocking for 1-3h at 4 ℃.
?. Microcentrifuge for 30 sec at 4℃. Wash pellet five times with 500ul cell lysis buffer. Keep on ice between washes.
?. Resuspend the pellet with 20ul SDS sample buffer. Vortex, then microcentrifuge for 30sec.
?. Heat the sample to 95-100℃ fro 5 min and microcentrifuge for 1min at 14000g.Or, do not heat.
?. Load the sample on a 4-20% gel for SDS-PAGE.
?. Analyze sample by western blotting.
TITLE: Access to Source Data/Documents (Part 10 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 10 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Product description and procedure summary</strong>:</p>
<p>Target cells are either selected or depleted by incubating your sample with the biotin anti-human CD14 antibody (clone 63D3) followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the CD14+ cells, do not discard them if those are your cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[GUIDELINES]
<p>This protocol has been optimized to remove washing steps after antibody and nanobeads incubation, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube from human peripheral blood mononuclear cells (PBMCs). If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p> </p>
<p>MojoSort™ Buffer (5X) (Cat. No. 480017)</p>
<p>MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system</p>
<p>Adjustable pipettes</p>
<p>70 μm filters (one per sample)</p>
<p>5 mL (12 x 75 mm) polystyrene tubes</p>
<p>Reagents for sample preparation</p>
<p>Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dseecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dseecje.jpg" data-ofn="mojosort human cd14.JPG" /></p>
<p><strong>Application notes:</strong> To use this product in magnetic separation columns, a titration of the cocktail/beads should be performed. Optimal concentration for magnetic separation columns is lot-specific.</p>
<p> </p>

[STEPS]
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TITLE: Treatment of DNA with cisplatin
AUTHORS: Martin O Pollard

[DESCRIPTION]
A protocol for treating Promega Human DNA with Cisplatin.

[GUIDELINES]
Dispose of excess cisplatin safely according to guidance received in SDS.

[STEPS]
SECTION: Treatment of DNA with Cisplatin
3. Pipette 8.811 µL of   from stock tube at 227 µg/ml to a 1.8 mL Eppendorf tube to achieve 2 µg of DNA.

SECTION: Treatment of DNA with Cisplatin
4. 
 
Add 7.27 µL of diluted cisplatin at 0.83 micromolar (µM) to the tube
SECTION: Treatment of DNA with Cisplatin
5. Incubate for 960 min in the dark at 37 °C.

SECTION: Preparation of Cisplatin liquid
1. 
 
Weigh out 25 mg of cisplatin into 1000 mL of 0.9% NaCl and mix thoroughly to make a stock solution.
Store stock at 2-8 °C and protected from light to avoid precipitation.

SECTION: Treatment of DNA with Cisplatin
6. Perform a SPRI bead clean-up (as previously described) to remove lingering cisplatin from supernatant.


SECTION: Treatment of DNA with Cisplatin
6.1. Add   of   SPRI beads to tube and 20% polyethylene glycol (PEG), 2.5 M NaCl buffer.

SECTION: Treatment of DNA with Cisplatin
6.2. Attach tube to magnet and remove supernatant.
SECTION: Treatment of DNA with Cisplatin
6.3. Resuspend DNA in 40 µL of buffer to achieve concentration of 50 ng/µl

SECTION: Preparation of Cisplatin liquid
2. Dilute 1 mL of your stock cisplatin solution into 99 mL of 0.9% NaCl and mix thoroughly.
Store diluted cisplatin at 2-8 °C and protected from light to avoid precipitation.
TITLE: Priming and loading a MinION flowcell
AUTHORS: Josh Quick

[STEPS]
?. Thaw the following reagents at room temperature before placing on ice:  Sequencing buffer (SQB)Loading beads (LB)Flush buffer (FLB)Flush tether (FLT)
?. Add  FLT to the FLB tube and mix well by vortexing.
30 µl
?. If required place a new MinION flowcell onto the MinION by flipping open the lip and pushing one end of the flowcell under the clip and pushing down gently.
?. Load  of FLB (plus FLT) into the flow cell via the inlet port, dispense slowly and smoothly trying to avoid the introduction of any air bubbles.
800 µl
?. Wait for.
?. In a new tube prepare the library dilution for sequencing:Component                                  VolumeSQB                                                LB                                                   Final library                                   Total
37.5 µl
25.5 µl
12 µl
75 µl
Mix LB immediately before use as they settle quickly. Dilute library in EB if required.
?. Mix the prepared library gently by pipetting up and down just prior to loading.
?. Add the  library dilution to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop siphons into the port before adding the next.
75 µl
?. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the inlet port and close the MinION lid.
?. Rotate the inlet port cover clockwise by 90° so that the priming port is visible.
?. Take a P1000 pipette and tip and set the volume to . Place the tip in the inlet port and holding perpendicularly to the plane of the flowell remove any air from the inlet port by turning the volume dial anti-clockwise.
800 µl
Be careful not to remove so much volume that air is introduced onto the rectangular array via the outlet.
?. Gently lift the SpotON cover to open the SpotON port.
?. Load another  of FLB (plus FLT) into the flow cell via the inlet port, this will initiate a siphon at the SpotON port to allow you to load the library dilution.
200 µl
TITLE: Study of MAIT Cell Activation in Viral Infections In Vivo
AUTHORS: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.</div></div>

[STEPS]
TITLE: Incidence and etiology of chronic pulmonary infections in patients with idiopathic pulmonary fibrosis
AUTHORS: Kyuto Odashima, Naho Kagiyama, Tetsu Kanauchi, Takashi Ishiguro, Noboru Takayanagi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Background</span><span>
The incidence and etiologies of chronic pulmonary infection (CPI) in patients with idiopathic pulmonary fibrosis (IPF) have been poorly investigated.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Methods</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">We conducted a retrospective study of 659 patients with IPF to assess the incidence, etiologies, and risk factors of CPI development. CPI was defined if the etiology of infection was diagnosed one or more months after the onset of symptoms or upon the appearance of new shadows on pulmonary radiological images.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Results</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>At IPF diagnosis, 36 (5.5%) patients had CPI, and 46 (7.0%) patients without CPI at IPF diagnosis developed CPI over a median follow-up period of 6.1 years. The incidence density of CPI development was 18.90 cases per 1000 person-years. Detected organisms from these 46 patients were </span><span style = "font-style:italic;">Mycobacterium avium </span><span>complex in 20 patients, othernontuberculous mycobacteria in 4, </span><span style = "font-style:italic;">M. tuberculosis</span><span> in 7</span><span style = "font-style:italic;">, Aspergillus</span><span> spp. in 22, and </span><span style = "font-style:italic;">Nocardia</span><span> sp. in one. In a multivariate Cox regression hazard model, PaO</span><span style = "vertical-align:sub;">2</span><span> <70 Torr and KL-6 ≥2000 U/mL were associated with CPI development.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Conclusions</span></div></div><div class = "text-block"><span>Nontuberculous mycobacteria, </span><span style = "font-style:italic;">M. tuberculosis</span><span>, and </span><span style = "font-style:italic;">Aspergillus</span><span>and </span><span style = "font-style:italic;">Nocardia</span><span> spp. were the four most frequent etiologies of CPI in patients with IPF. During follow-up of IPF, clinicians should pay attention to the development of CPI, especially in patients with PaO</span><span style = "vertical-align:sub;">2</span><span> <70 Torr or KL-6 ≥2000 U/mL.</span></div></div>

[STEPS]
TITLE: Nuclear extraction from endometrial tumors for single nuclei sequencing 
AUTHORS: gracefoley  

[DESCRIPTION]

Modified from: Slyper, M. et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat. Med. 2020 26526, 792–802 (2020).

A optimized protocol for nuclear extraction from endometrial tumors.  Performed with endometrial adenocarcinoma, endometrioid type, FIGO grade 1.  

  


[STEPS]
1. Place frozen tissue in a well of a 6 well plate containing 1 mL of cold NST disassociation buffer.
2. Chop tissue using a scalpel until mostly homogenized
3. Incubate solution on ice for 5 minutes
4. Filter the homogenized solution through a 40 uM falcon cell strainer into a 50 mL falcon tube
5. Use an additional 1 mL of NST buffer to rinse the well and filter
6. Count nuclei
7. Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
8. Resuspend pellet and bring the volume up to 5 mL using ST buffer
9. Transfer solution to 15 mL conical tube and centrifuge for 10 minutes at 300 g at 4oC
10. 10.  Resuspend pellets on ice in 1 ml of the ST buffer
11. 11.  Filter through 35 um falcon strainer
12. 12.  Count nuclei
14. 13.  Resuspend nuclei pellet in resuspension buffer to get a concentration of 1,000 nuclei per uL
13. Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
14.1.     Start with .5 mL per 0.2 grams of endometrial tumor tissue
14.2. Look at nuclear membranes at 40x-60x to ensure high nuclear membrane quality with minimal blebbing
1.1. Ensure plate is on ice
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to wash QCM-D sensors before usage and how to modify their surface using a composition of symmetric (poly)ethylene glycol (PEG) thiols consisting of 99% dS-PEG and 1% dS-PEG-biotin.

[BEFORE_START]
<br /><br /><br /><br /><br /><br /><br /><br /><br />

[STEPS]
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TITLE: Via Keto Capsules FR Reviews, Side Effects, Ingredients 2022
AUTHORS: viaketocapsulesreviews  

[DESCRIPTION]
Via Keto Capsules FR Reviews, Side Effects, Ingredients 2022


[STEPS]
1. Via Keto Capsules FR Reviews, Side Effects, Ingredients 2022
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TITLE: Protocol for Exonuclease III (NEB #M0206)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Exonuclease III efficiently degrades nicked and linear dsDNA (with blunt or 5' overhangs) from 3' to 5' direction, leaving supercoiled dsDNA intact.*</div><div class = "text-block"><span style = "font-weight:bold;">*</span><span style = "font-weight:bold;">Note: </span><span>For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.</span></div></div>

[STEPS]
?. Set-up the reaction as follows:  AB1COMPONENTS50&nbsp;μl REACTION2&nbsp;DNA&nbsp;up to 5 μg3&nbsp;NEBuffer 1 (10x)&nbsp;5 μl (1X)4&nbsp;Exonuclease III&nbsp;0.5 μl (50 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
AB1COMPONENTS50&nbsp;μl REACTION2&nbsp;DNA&nbsp;up to 5 μg3&nbsp;NEBuffer 1 (10x)&nbsp;5 μl (1X)4&nbsp;Exonuclease III&nbsp;0.5 μl (50 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
?. Incubate at  for .
37 °C
?. Stop reaction by adding EDTA to at least .
?. Heat inactivate at  for .
70 °C
?. To clean up treated samples, we recommend using one of the following steps:a. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030), orb. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), orc. Performing a phenol/chloroform extraction followed by ethanol precipitation.