eamag/protocols-with-steps huggingface dataset:
TITLE: Determination of MIC according to the EUCAST E.DEF 11.0 method
AUTHORS: Khalid  El Moussaoui

[DESCRIPTION]
Determination of MIC according to the EUCAST E.DEF 11.0 method.

[STEPS]
1. With the disinfected tweezers, remove the filter (attention: do not take the filters of protections having a slightly bluer tint) and place them in the support provided for this purpose. Close the holder.
2. Pour the contents of TUBE A (sterile water + Tween) onto the Petri dish containing the starting sample and wipe with a swab until the suspension is relatively cloudy.
3. Aspirate the suspension with the syringe.
4. Filter this suspension by injecting it into the filter (0,45 µm) holder and collect the filtered suspension in
suspension in TUBE A.
5. Measure the density of TUBE A and adjust it to 0.5 McFarland. Then take 500 μL of this
suspension and place it in TUBE B (to be done 2x since we are working in duplicates). TUBE B contains sterile demineralized water (Thermo Fisher T3339).
6. Place an inoculation head on the tube and inoculate with the Sensititre Aim.
7. Incubate at room temperature for 7 days and read the result at 492 nm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>A real-time RT-PCR targeting the 5' untranslated region of Yellow fever virus.</p>
<p>This protocol was designed and developed at this laboratory.</p>
</div>

[GUIDELINES]
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<strong>Purpose:</strong> to determine the concentration of DNA-containing particles in a given sample; useful in finding cell counts or phage titers.<br /><br />

[GUIDELINES]
<strong>Materials:<br /></strong><br />- &nbsp; 0.02 &micro;m pore size, 25 mm diameter Anodisc filters (Whatman inorganic Al<sub>2</sub>O<sub>3</sub>)<br /><br />- &nbsp; 0.8&nbsp;&micro;m pore size, 25 mm diameter AA Millipore mixed-ester membrane filters<br /><br />- &nbsp; Glass 25 mm diameter filter holder w/ 15 mL funnel, Millipore-type<br /><br />- &nbsp; 10% SYBR I solution from Molecular Probes, diluted with 0.02 &micro;m&nbsp;filtered deionized water. Make sure to wrap the stock in aluminum foil and to keep it in the dark.<br /><em>As time passed the SYBR stock should degrade, acquiring a more reddish color while frozen and a a paler orange color after being thawed. This can initially be addressed by doubling the amount of SYBR used, but it is a sign that the stock should soon be replaced.<br /></em><br />- &nbsp; Antifade mounting solution: 50% glycerol, 50% PBS (120 mM salt, 10 mM sodium phosphate, pH 7.5), 0.1% phenylenediamine (Sigma cat#P6001).<br /><em>Original protocol says to add the phenylenediamine from a 10% stock; however the maximum solubility of the free base is only 4.1 g/mL (the 10% concentration can be prepared by adding HCl to make the chloride salt). To make the stock, dissolve the powder in filtered deionized water by vortexing and adding HCl as needed; wrap in aluminum foil and stare at -20&deg;C. Stock is light and temperature sensitive, and will go darker with time, corresponding to a decline in quality. A fresh free base solution is pink-orange while the chloride salt is purple. Use of antifade made with poor (e.g. brown or black) phenylenediamine may result in slides with particles that appear faint or seem to fade with time; with a brown stock this may be corrected by using a higher (e.g. 5x) concentration of phenylenediamine.&nbsp;</em><br /><em>An alternative (which we have been recently doing) is making a 1% stock of the free base dissolved in 50% PBS, 50% glycerol. A fresh stock of this should be pinkish in color. The advantage of using this is several-fold: first, adding concentrated acid is no longer necessary; second, because the stock is more dilute it should be used up faster, so that it will need to be remade more often and thus remain fresher; third because it is 50% glycerol it does not freeze at -20 thus saving thawing time.</em><br /><br />- &nbsp; Sample, autoclaved seawater, Petri dish, pipettes and tips, dessicator, kimwipes, syringe, and 0.02 &micro;m 25 mm Anotop filters.<br /><br /><strong>Source:</strong> from the original protocol (July 2001), adapted to working with cyanophages.&nbsp;<br />Noble, T.T., and J.A. Fuhrman. 1998. "Use of SYBR Green I for Rapid Epifluorescence Counts of Marine Viruses and Bacteria." Aquatic Microbial Ecology. 14: 113-118.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time RT-PCR using an MGB probe, this assay detects Japanese encephalitis virus (JEV) from human and mosquito samples.</p>
<p>The assay targets the 3'UTR region of known JEV strains.</p>

[BEFORE_START]
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check that the concentration of ROX is adequate.</li>
<li>Method assumes the user is familar with the thermocycler and software used to run the protocol.</li>
</ul>
</div>

[GUIDELINES]
<p>The concentration of ROX in this method is for the ABI7500 real-time thermocycler. It is not necessary for a Rotor-gene, however we run the assay with it in the recipe on a Rotor-gene machine. Concentration should be adjusted if using a different machine. Check your manufacturers' manual for guidance.</p>

[STEPS]
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TITLE: Mitomycin C stem cell ablation
AUTHORS: Simon Blanchoud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Treatment is done for 24 h right after WBR 〈Whole Body Regeneration〉 induction. Tissue will not regenerate but haemolymph flow should be observable for around 20 days. A number of samples have died following this treatment, hence multiple pieces of tissue were treated per slide.</div></div>

[STEPS]
?. [MMC solution]
Dissolve  of MMC in  of dH2O.
1 mg
500 µl
?. [MMC solution]
This is the 1000x stock solution 〈  ).
?. [MMC solution]
Aliquot this volume (we did it in  ) and store at  .
100 µl
-20 °C
?. [MMC solution]
Mix  of MMC stock solution with  of FSW.
10 µl
990 µl
?. [MMC solution]
This is the working solution ( ).
?. [MMC treatment]
Induce WBR in at least two portions of the colony.
?. [MMC treatment]
Gently dry around the edges of the regenerating pieces of tissue.
?. [MMC treatment]
Dry the whole slide as much as possible.
?. [MMC treatment]
Circle the regenerating pieces of tissue using the Pap pen.
?. [MMC treatment]
Wait for the liquid to fully dry (~  ).
?. [MMC treatment]
Cover one tissue with as much FSW as possible (>  ) as control.
200 µl
?. [MMC treatment]
Cover the other pieces of tissue with as much MMC solution as possible.
?. [MMC treatment]
Place the slide horizontally in the humidity chamber.
?. [MMC treatment]
Place the chamber at a temperature close to their culture temperature.
?. [MMC treatment]
Leave the slide to incubate for  .
?. [MMC treatment]
Rinse the slide in FSW.
?. [MMC treatment]
Clean the Pap pen circles using a wet Q-tips.
?. [MMC treatment]
Place the slide back in the aquarium.
?. [MMC treatment]
Monitor daily.
TITLE: OpenVent Eco DNA Polymerase Production
AUTHORS: Jenny Molloy, Stephane Fadanka, Nadine             Mowoh

[DESCRIPTION]
This collection contains the protocols for production, quality control, packaging and use of Beneficial Bio's dehydrated OpenVent DNA Polymerase.

Format: 32 tubes (4x8-tube PCR strips) 20ul of Enzyme each

OpenVent DNA polymerase is an extremely high thermostable and high fidelity enzyme suitable for routine PCR applications and amplification of GC-rich or looped sequences. The enzyme is compatible with a wide range of templates and its robustness guarantees reliable amplification results in almost all PCR applications. The Eco and room temperature stable format - PCR reaction tube strips containing the dehydrated enzyme (10 reactions per tube), offers affordability and flexibility.

FEATURES
Dehydrated formulation enables better stability at room temperature
Ideal for a wide range of applications including GC-rich or looped sequences amplification
Amplification of low copy DNA targets
Eco formulation and flexible format

DOCUMENTS

Product Manual
Product Specification Sheet (PSS)
Certificate of Analysis (CoA)

PROTOCOL GUIDANCE

Select one of the following options for enzyme production based on your lab infrastructure:
IPTG-based induction requires a shaking incubator, large benchtop centrifuge and IPTG
Plate-based autoinduction requires none of the above

After producing the enzymes, perform the three QC protocols in any order:
SDS-PAGE to determine expression has been successful
Functionality testing in PCR
Nuclease contamination testing




 
 

[STEPS]
TITLE: Evercode Single Index PCR
AUTHORS: Parse Biosciences, Elisabeth Rebboah

[DESCRIPTION]
This protocol describes the single-index PCR procedure for Parse Biosciences Evercode WT and WT Mega v2 kits. Each subpool is barcoded with a single Illumina index on the 3' end of the cDNA library. This acts as the fourth "round" of cell barcoding and must be included in the final cell ID/barcode in order to ensure unique barcodes across subpools within an experiment. The numerical ID and sequence of the Illumina barcode used for each subpool are recorded in experiment metadata and used downstream for subpool demultiplexing after the sequencing run.

[STEPS]
SECTION: Appendix D2: Sublibrary Single Index PCR
1. If using unique dual indexes (UDIs) instead of sublibrary single index primers for indexing, see Section 3.5. Otherwise, replace the entirety of Section 3.5 with the following steps.

Multiple thermocyclers may be needed for this section depending on the amount of cDNA added to each sublibrary during the fragmentation reaction. Refer to step next page to determine how many thermocyclers are needed.
SECTION: Appendix D2: Sublibrary Single Index PCR
2. Using a new 1.5 mL tube, combine the Universal Index Primer and Index Primer Mix to make the Sublibrary Amplification Mix. Mix well by pipetting and store on ice.


 

SECTION: Appendix D2: Sublibrary Single Index PCR
3. Add 2 μL of different index primers to each sublibrary ensuring that no two sublibraries contain the same sublibrary index primer. Make sure to record which sublibrary contains which index primer.
SECTION: Appendix D2: Sublibrary Single Index PCR
4. Add 27 μL Sublibrary Amplification Mix to the 23 μL sublibrary from the previous step. Pipette up and down 10x with the pipette set to 27 μL to ensure proper mixing, followed by brief centrifugation (~2 sec).
SECTION: Appendix D2: Sublibrary Single Index PCR
5. Place the samples(s) into a thermocycler and run the program below. The number of cycles (X) should be adjusted based on the amount of cDNA added to the fragmentation reaction.


 


 


 


Note: cDNA concentration was recorded in step 2.5.18, and 10 μL from each sublibrary should have been added into the fragmentation reaction (step 3.1.2).
SECTION: Appendix D2: Sublibrary Single Index PCR
6. Sublibraries can be stored at this point at 4°C overnight. If you wish to continue, proceed directly to Section 3.6: Post-Amplification Double-Sided Size Selection.
[STOPPING POINT]
TITLE: Agarose Gel Electrophoresis (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for
 

The protocol is pretty straightforward, but I have included several common student errors to watch out for!

[STEPS]
SECTION: Make the LAB buffer
1. Make LAB buffer following this recipe:
 

For a class of 24, I usually prepare 6x 1 L bottles of 1X LAB. We go through a lot -- there are three gels that get run over the course of the semester, plus repeats.

SECTION: Instructor Tips &amp; Common Student Errors
2. Instructor Tips
The benefit of using LAB is the ability to run gels at higher voltage -- our "mini" gel tanks go at 120 V for 20 minutes and get pretty decent gels. It is totally possible to cast, load and run an LAB gel in 2 hours.
I usually demonstrate the proper assembly of the gel tank
Gels with Sybr Safe are fine in the refrigerator for 48 hours, but much longer than that and the stain degrades too much. That is -- a gel cast on Tuesday will be fine for a lab on Thursday, but not the other way around. To store a gel, put it in a zipper sandwich baggie and add a splash (not more) of LAB buffer. Label and store at 4 °Cin the refrigerator.
A common student error is to forget the DNA stain. But all is not lost -- you can post-stain these gels. Put the gel in a small container (a tip box lid is often fine), cover with LAB, add 5 µL of Sybr Safe, and put on a slow rocker or orbital shaker.

SECTION: Instructor Tips &amp; Common Student Errors
3. Common Student Errors
Forgot to add the DNA stain
Didn't 100% dissolve the agarose (it should be CLEAR AS WATER, no flecks floating around in it)
Ran the gel backward (remember, "run to red")
Only ran 1 µL of the restriction digest (instead of the whole digest)
Alternately, ran the entire PCR (instead of just 1 µL) -- leaving not enough to purify.
TITLE: Prepare Uric Acid Solution
AUTHORS: 宏亮 董

[STEPS]
?. Prepare Uric Acid Storage Solution:Accurately weigh 0.01g uric acid dissolved in Uric Acid Diluent Solution and dilute to 100mL;
?. Prepare Borate Buffer Storage Solution:Add these reagents to a clean media bottle, DDW volume to 200ml, adjust pH to 8.5 with NaOH.  AB1boric acid6.185g2borax9.535g3EDTA1.169g410% Triton X-100400μl
AB1boric acid6.185g2borax9.535g3EDTA1.169g410% Triton X-100400μl
?. Prepare Uric Acid Diluent Solution:Add 152 mL DDW and 8 mL Borate Buffer Storage Solution to a clean media bottle, adjust pH to 8.5 with NaOH.
?. Prepare Uric Acid Working Solution:Dilute Uric Acid Storage Solution with Uric Acid Diluent Solution for 10 times. The concentration can be adjusted according to experimental requirements.
TITLE: KAPP-Sen TMC: Preparation of Workspace for Tissue Blocks Sectioning
AUTHORS: Juliana Alcoforado Diniz, Elaine Bechtel, Paul Robson

[DESCRIPTION]
Preparation of Workspace for Tissue Blocks Sectioning

[STEPS]
SECTION: Preparation of Workspace
1. Wear nitrile gloves cleaned with RNase Zap wipe.

SECTION: Preparation of Workspace
2. Using RNase Zap wipes, clean microtome including any surface block or hands may contact (block holder, knife holder, handle, etc.).
SECTION: Preparation of Workspace
3. Using RNase Zap wipes, clean glass Pyrex water bath insert and rinse with deionized water. Fill with deionized water and heat per usual.
SECTION: Preparation of Workspace
4. For ice tray, using RNase Zap wipes, clean ice tray. For ice block, at least 24hrs prior to sectioning, using RNase Zap wipes, clean plastic ice container and rinse with deionized water. Fill with deionized water and put in -20°C freezer. To use, cover ice with enough deionized water that the blocks will not freeze to ice.
SECTION: Preparation of Workspace
5. Using RNase Zap wipes, clean forceps and brushes/applicator sticks.
**This step will need to be repeated between each block.**
SECTION: Preparation of Workspace
6. Using RNase Zap wipes, clean slide racks that will hold unstained slides.
TITLE: Risk perception and coping response to COVID-19 is mediated by positive and negative emotions: a study on Chinese college students
AUTHORS: ganyt  

[DESCRIPTION]
This study aimed to assess the mediating roles of positive and negative emotions on the relationship between COVID-19-related risk perception and coping behaviours adopted by Chinese college students in response to the COVID-19 pandemic. We conducted an internet-based questionnaire survey from mid February–late October 2020, among 1038 college students, from six Chinese universities (females = 73.41%), ranging within 17–26 years. The survey questionnaire included three major components—the COVID-19-Related Risk Perception Scale (CRPS), the Positive and Negative Affect Scale (PANAS-Revision), and Coping Response of COVID-19 Scale (CRCS). Descriptive statistics and a mediated model were used to analyse the collected data.A partial mediation relationship was found between COVID-19-related risk perception and 1) active-response behaviour (β=0.05, 95% Confidence Interval [CI: 0.03, 0.08]), 2) self-protection behaviour through positive emotions (β=0.03, CI [0.01, 0.04]), and 3) risk-taking behaviour through negative emotions (β=-0.04, CI [-0.07, -0.02]). This study’s double-mediation model has been shown to detect the effect coping mechanisms to COVID-19. Furthermore, it implies that public health managers should consider the differences in coping mechanisms and the diverse mediating roles of positive and negative emotions for coping with public health emergencies. 

[STEPS]
TITLE: Treatment of D. melanogaster with small molecules in fly food
AUTHORS: Natalie Kaempf, Patrik Verstreken

[DESCRIPTION]
This protocol describes the preparation of fly food with small molecules to treat flies during their aging. It can be adapted for any treatments. Details for small molecules used can be found in the "Materials" section

[STEPS]
SECTION: Food preparation
1. warm up the amount of fly food (standard corn meal and molasses food) required (e.g. 50ml for 45 XS vials) in the microwave, stir it in between, start with 1.5 min, mix the food, heat up for 1min again and continue until food has no clumbs anymore 
SECTION: Food preparation
2. prepare XS vials (45 for each condition when 50ml/condition) and label them, place them in plastic boxes 
SECTION: Food preparation
3. measure the amount of food in measuring cylinder and let it cool down in the beaker to around40 °C (immediately clean all glass ware after usage)

SECTION: Food preparation
4. add compound/solvent and stir very well (1 min ), to keep the food liquid put in water bath to maintain the temperature
SECTION: Food preparation
5. pipette around 1 mL per XS vial with 10 or 25 ml pipette

SECTION: Food preparation
6. Make sure the food is filling the whole bottom of the vial and shows even surface to avoid flies getting stuck in small wholes when aging
SECTION: Food preparation
7. store the food in darkness at 4 °C in the cold room and clean up

SECTION: Food preparation
8. use the food not longer than 2 weeks
SECTION: fly treatment
9. depending on the experimental condition flies are exposed to the treatment and corresponding control after they are collected (Q10 or R55 treatment) or 10 days prior the experiment (D-/L-DOPA treatment) 
SECTION: fly treatment
11. flies are flipped to new food every two to three days
SECTION: fly treatment
12. On the day of the experiment flies are flipped on fresh treatment/control food at Zeitgeber zero to one
SECTION: fly treatment
10. flies on treatment/control food are kept in a temperature and light-controlled incubator at
25°C and 12-hour light-dark cycle
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The present protocol explains how to prepare a primary culture of isolated neurons from the mouse nodose ganglion.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are your cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[GUIDELINES]
<p><strong>Reagents and instruments required:</strong></p>
<p> </p>
<p>-MojoSort™ Buffer (5X) (Cat. No. 480017)<br />-MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />-Adjustable pipettes<br />-70 μm filters (one per sample)<br />-5 mL (12 x 75 mm) polystyrene tubes<br />-Reagents for sample preparation<br />-Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p><br /><strong>Protocol:</strong></p>
<p>This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 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><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>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dn6ecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dn5ecje.jpg" data-ofn="MojoSort_isolationkitsreg.JPG" /></p>

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TITLE: Comparative efficacy of sedation or analgesia methods for reduction of anterior shoulder dislocation: a systematic review and network meta-analysis
AUTHORS: Minoru  Hayashi, Kenichi Kano, Naoto Kuroda, Norio Yamamoto, Akihiro Shiroshita, Yuki Kataoka

[DESCRIPTION]
The objectives are as follows:
To review systematically comparative efficacy of sedation or analgesia methods for reduction of anterior shoulder dislocation.

[STEPS]
TITLE: Proximity Ligation Assay (PLA)
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Proximity Ligation Assay (PLA)

[STEPS]
1. The PLA assay was performed as described previously with minor modifications 48. The following antibodies were used for the PLA experiments: Syn 204 against h-αSyn (Santa Cruz Biotechnology Cat# sc-32280, RRID:AB_628319)(1:100) and EPR12790 against VAMP-2 (Abcam Cat#
ab214590)(1:300). 




2. The in-situ PLA was performed on fixed primary neurons
with DuoLink PLA technology probes and reagents (Sigma-Aldrich Cat# DUO92002,
DUO92004, DUO82049, DUO92008, and DUO92014), following the manufacturer’s
protocol. First, the neurons were permeabilized with PBS + 0.4% Triton X-100
for 10 min. 


3. After two PBS washes, the cells were incubated with a blocking
solution for 2 hours at 37 °C and then incubated with the primary antibodies
for 30 min at room temperature. 
4. The coverslips were washed twice for 5 min with
buffer A, followed by incubation with the PLA probes (secondary antibodies
against two different species bound to two oligonucleotides: anti-mouse MINUS (Sigma-Aldrich Cat#
DUO92004 (also DUO92004-30RXN, DUO92004-100RXN), RRID:AB_2713942) and anti-rabbit PLUS) (Bethyl Cat# OLK-92002-0100,
RRID:AB_10950581)in antibody diluent for 30 min at 37 °C. After two washes of 5
min with buffer A, the ligation step was performed with ligase diluted in
ligation stock for 30 min at 37 °C. 
5. The coverslips were washed with buffer A
twice for 2 min before incubation for 50 min with amplification stock solution
at 37 °C. After two washes of 10 min with buffer B. Finally, the coverslips
were washed with PBS and mounted with Duolink in situ mounting medium
(Sigma-Aldrich Cat# DUO82040-5 ML). 
6. A negative control experiment was performed
for every antibody, where only one antibody was incubated with the PLA probes.
The experiments were performed 2 times. The experiments were performed 2 times.
Average signal intensities were measured using the  MetaMorph Microscopy Automation and Image
Analysis Software (RRID:SCR_002368) (https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref) and plotted using GraphPad Prism
software [(RRID:SCR_002798) http://www.graphpad.com/].