eamag/protocols-with-steps huggingface dataset:
TITLE: Minimum inhibitory concentration of butanol for E. coli KJK01
AUTHORS: Arshshaikh  

[DESCRIPTION]
This protocol helps you determine the minimum inhibitory concentration (MIC) of butanol for E. coli KJK01. However, this technique can be extended to determine the MIC of any metabolite for any strain by making adjustments to the concentration of the metabolite in the starting wells.

[STEPS]
1. Take a 96 welled plate.

SECTION: Media condition preparation
2. Calculate the amount of butanol that you require in the starting well. Wilbanks 2017 paper suggests that butanol shows strong toxic effects below 10 g/L and entirely inhibits growth at 15 g/L. We should expect our MIC to fall in this range. Hence, we can start with an initial concentration of 60 mg/ml. 
 

SECTION: Media condition preparation
2.1. Take 185.2 µL  and pour it in wells B2 to D2.

SECTION: Media condition preparation
3. Pour 100 µL of LB in all wells from B3 to B11 and the same for rows C and D.

SECTION: Media condition preparation
4. Take 14.6 µL  and pour it wells B2 to D2 and mix well.

SECTION: Media condition preparation
5. Take 100 µL and pour it in well B3. Mix well.

SECTION: Media condition preparation
6. Take 100 µL and pour it in well B4. Mix well. Repeat this for the subsequent wells till you reach well B10.
SECTION: Media condition preparation
7. Take 100 µL  and discard in the well B12. 
 


SECTION: Media condition preparation
8. Repeat steps 5 to 7 for rows C and 
SECTION: Inoculation
9. Take 0.2 µL  and inoculate wells B2 to B11.
SECTION: Measurement
13. Cover the 96 welled plate with foil and place it in the incubator at 37oC.
SECTION: Inoculation
12. Fill the border wells with milliQ.

SECTION: Measurement
14. Take a reading of the plate using any plate reader every hour and place it back in the incubator. You may run this for 24 hrs.
SECTION: Measurement
15. Obtain a growth curve for each strain and concentration. Identify the least concentration for which the growth curve shows a sharp drop. 
SECTION: Inoculation
10. Take 0.2 µL  and inoculate wells C2 to C11.
SECTION: Inoculation
11. Take 0.2 µL  and inoculate wells D2 to D11.
TITLE: E. coli  growth trend measurement (culture with silver ion)
AUTHORS: Weizhuo.Chen, An.Huang

[DESCRIPTION]
This protocol helps obtain growth curve of E. coli cultured with concentration gradient of silver ion.

[STEPS]
SECTION: IPTG induction
1. Grow Escherichia coli overnight and dilute the culture using LB to OD600=0.4-0.6.
SECTION: IPTG induction
2. Add IPTG to cell culture (final concentration 1mM), and incubate the culture 3h at 171 rpm, 37℃ in orbital shaking incubator.
SECTION: Sample addition to microplate wells
3. Cells cultures, LB broth and silver nitrate (150μM) are added into 96-well microplate. The recipe is as the following chart:
  

SECTION: Microplate reader measurement
4. Incubate the microplate in orbital incubator (37℃ 171 rpm) and measure OD600 by microplate reader every two hours for two days.
TITLE: Nile Red Staining of Drosophila Larval Tissues
AUTHORS: Elizabeth Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to stain late larval </span><span style = "font-style:italic;">Drosophila</span><span> lipid droplets in fat bodies and intestines with Nile Red, which emits fluorescence in the 552/636 nm range. </span></div></div>

[STEPS]
?. Dissect tissues in ice cold PBS, keeping samples on ice until required sample size is obtained.
?. Fix tissues in 4% PFA for 20 minutes.
?. Wash tissues 3x in PBS.
?. Stain samples in Nile Red at 0.5 ug/mL diluted in PBS for 1 hour.
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?. Wash tissues 3x in PBS.
?. Carefully replace the PBS with mounting medium before transferring samples to slides and imaging. * Image immediately, or temporarily store slides at 4°C.
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TITLE: LYSOSOMAL ISOLATION PROTOCOL
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the isolation of lysosomes.

[STEPS]
SECTION: Subcellular Lysosome Isolation Protocol
1. In Vitro Harvest
SECTION: Subcellular Lysosome Isolation Protocol
1.1. Following designated treatment, wash cells three times with DMEM followed by the application of 0.5 mL of homogenization buffer supplemented with proteinase and phosphatase buffer.

 Homogenisation Buffer:
 
 
SECTION: Subcellular Lysosome Isolation Protocol
1.2. Gently detach cells with cell scraper.

SECTION: Subcellular Lysosome Isolation Protocol
1.3. Homogenize using Teflon homogenizer (12 strokes).  

SECTION: Subcellular Lysosome Isolation Protocol
1.4. Separate 50 µL of total homogenate (TH) and lyse with *TNE lysis buffer. 

 TNE lysis buffer:
 
 
SECTION: Subcellular Lysosome Isolation Protocol
1.5. Centrifuge remaining volume of TH at  1000 x g, 10 min, 4 °C, collect the supernatant. 

SECTION: Subcellular Lysosome Isolation Protocol
1.6. Further centrifuge the supernatant for 20000 x g, 20 min, 4 °C to collect the precipitate as crude lysosomal fraction (CLF). Lyse the CLF with *TNE lysis buffer, and prepare lysates for western blot.

 TNE lysis buffer:
TITLE: Tissue Protein Extraction: Tissue Homogenization using Urea-based Buffer and Bead Mill Homogenizers
AUTHORS: J Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Tissue homogenization to isolate protein in preparation for downstream proteomic profiling. 

[STEPS]
9. Transfer the supernatant (clear lysate containing the soluble proteins) to a clean
1.5-mL microcentrifuge tube, carefully avoiding any liquid fat layer on top and
debris at the bottom of the tube.
1. Chill the adaptor sets of the bead mill homogenizer at -20°C.


2. Freshly prepare the lysis buffer as described in Table 1 and keep cold.

 


3. On dry ice, add the tissue specimen to a 2-mL microcentrifuge tube compatible with the bead mill homogenizer, then place the metallic bead into the tube. Add 500 µL of cold lysis buffer to the tissue specimen (add more lysis buffer to cover the tissue specimen, if necessary).

4. Place the tubes in the prechilled homogenizer adaptor sets, ensuring that the tubes are balanced between the two adaptors.
5. Homogenize the samples for 2 cycles at 24 Hz for 2 min each. If the tissue specimen is not fully homogenized, spin the samples briefly, transfer the homogenized lysate into a clean 1.5-mL microcentrifuge tube, add additional lysis buffer, and repeat the homogenization step. Two cycles of homogenization are typically enough to break up the tissue.

6. Remove the bead with a tweezer. In between each sample, rinse the tweezer with milliQ water, then HPLC-grade methanol, and dry thoroughly with a delicate task wiper.
7. Transfer the homogenized lysate into a clean 1.5-mL microcentrifuge tube or combine the homogenized lysates if needed, carefully avoiding bubbles and the separated fat layer at the top of the supernatant.
8. Centrifuge the homogenized lysate at 15,700 x g for 15 min at 4°C to clear the lysate.

10. Perform a bicinchoninic acid (BCA) protein assay to determine the protein concentration of the clear lysate with a proper dilution (typically 1:20 - 1:200 depending on the tissue type). According to the BCA results, transfer an aliquot of the sample into a clean 2-mL microcentrifuge tube for subsequent proteolytic digestion.
TITLE: Use of drugs associated with QT interval prolongation at the hospital level during the start of the COVID-19 pandemic in Colombia
AUTHORS: Jorge        Machado Alba, Andrés Gaviria-Mendoza, Manuel Enrique Machado-Duque, Luis Fernando Valladales-Restrepo

[DESCRIPTION]
Background: Many of the therapeutic proposals for COVID-19 have been associated with adverse effects, including the risk of QT interval prolongation and torsades de pointes (TdP). The objective wasto determine the use of drugs with a risk of QT interval prolongation in 21 clinics/hospitals in Colombia from January to December 2020.
Methods: This Cross-sectional study identified drug use according to pharmacological groups with potential risk of QT interval prolongation according to a risk classification: conditional, possible and known risk of TdP. Descriptive analyses were performed.
Results: A total of 355,574 patients who received QT-prolonging drugs were identified (equivalent to 51.4% of all inpatients treated during the study period). Of the group of patients on QT drugs, 54.4% used at least one drug with conditional risk, 52.6% with possible risk and 40.3% with known risk. The most commonly used belonged to the group of drugs for the nervous system (63.0%), alimentary tract and metabolism (56.8%), anti-infectives for systemic use (13.0%) and the cardiovascular system (11.7%). On average, patients received 2.0 ± 1.5 risk drugs. Regarding drugs initially considered against COVID-19, 2,120 patients (0.6%) received azithromycin, 802 (0.2%) received chloroquine, 517 received hydroxychloroquine (0.1%) and 265 received lopinavir/ritonavir (0.1%).
Conclusion: The high proportion of patients treated at the hospital level who receive drugs with risk of prolonging the QT interval should alert those responsible for their care to avoid fatal outcomes, especially during the COVID-19 epidemic, when some QT drugs are being used more frequently.

[STEPS]
TITLE: The combination of LMWH (Low-Molecular-Weight Heparin) and Aspirin as a prophylaxis treatment for pre-eclampsia and SGA (Small-for-Gestational Age) neonates in medium- and high-risk women: protocol for a Systematic Review and Meta-Analysis.
AUTHORS: Christos-Georgios Kontovazainitis, Georgios Mitsiakos, Dimitra Gialamprinou

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol to generate axenic fly cultures by sterilizing embryos. It is part of the manuscript: <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></p>
<p> </p>
<p>The protocol was adapted from:</p>
<p> </p>
<p>Wayland MT, Defaye A, Rocha J, Jayaram SA, Royet J, Miguel-Aliaga I, et al. Spotting the differences: Probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila. J Insect Physiol. 2014;69: 126–135. doi:10.1016/j.jinsphys.2014.05.023</p>
<p> </p>
<p>with the help of the Leulier laboratory.</p>
<p> </p>
<p> </p>

[BEFORE_START]
<p>Prepare the required fly media.</p>

[GUIDELINES]
<p> <img src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-ofn="Screen Shot 2017-03-18 at 1.55.25 AM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Prepare high yeast-based medium (HYBM) as follows:</p>
<ul style="text-align: justify;">
<li>add 8 g agar, 80 g barley malt syrup, 22 g sugar beet syrup, 80 g corn flour, 10 g soya flour, <strong>41.67 g instant yeast</strong>, 8 ml propionic acid, 12 ml nipagin (15% in 96% ethanol), <strong>416.7 µg/ml tetracycline (high dose), 41.67 µg/ml chloramphenicol, 41.67 µg/ml ampicillin, and 8.333 µg/ml erythromycin</strong> and make up to 1000 ml of milliQ filtered water</li>
<li>autoclave before pouring into polypropylene fly culture vials (VWR, #734-2261)</li>
<li><strong>DO NOT supplement the food with instant yeast granules on the surface</strong></li>
</ul>
<p> </p>
<p>The high yeast concentration is to compensate for the developmental delay observed in axenic larvae (Storelli G, Defaye A, Erkosar B, Hols P, Royet J, Leulier F. Lactobacillus plantarum Promotes Drosophila Systemic Growth by Modulating Hormonal Signals through TOR-Dependent Nutrient Sensing. Cell Metab. 2011;14: 403–414. doi:<a href="http://dx.doi.org/10.1016/j.cmet.2011.07.012" target="_blank">10.1016/j.cmet.2011.07.012</a>).</p>
<p> </p>
<p> </p>
<p>Perform all fly rearing, maintenance, and behavioral testing at 25°C in climate-controlled chambers at 70% relative humidity in a 12-hr-light-dark cycle (Aralab, FitoClima 60000EH).</p>

[STEPS]
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TITLE: Histology and Retention of Implanted Carbon Fiber Thread Electrodes in Fixed Tissue    
AUTHORS: Helen N Schwerdt, Tomoko Yoshida, Ann M Graybiel

[DESCRIPTION]
Methods to cut and retain implanted carbon fiber electrodes in brain tissue are described.

[STEPS]
1. Rats were deeply anesthetized with Euthasol (pentobarbital sodium and phenytoin sodium from Virbac AH Inc.), then transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB).
2. The implanted electrodes were cut just above the skull and below the copper wire bundle component of the device with a rotary saw (Dremel) to retain the flexible Py-CF portion of the device in the fixed brain tissue.
3. Brains were removed from the skull, post-fixed for 24 hours in the same fixative solution and stored in 25% glycerol with 0.075% sodium azide in 0.1 M PB overnight or until cutting.
4. To prepare sections, brains were frozen on dry ice then cut in the transverse plane on a freezing microtome with a microtome knife (C. L. Sturkey, Inc.  #K185A).
5. Sections were cut sequentially, with four sections at 25-μm then one section at 100-μm, repeated until the end of the frozen block of brain.
6. The 100-μm sections were immediately mounted onto glass slides and air dried, whereas the 25-μm sections were stored in 0.1% sodium azide in 0.1 M PB at 4°C until use.
TITLE: Visually-Guided Reward-Biased Behavioral Task
AUTHORS: Raymond Murray, Helen Schwerdt

[DESCRIPTION]
A protocol describing the visually guided, reward-biased saccade task monkeys were trained on in the original study: Schwerdt et al. 2020, Sci. Adv. 

[STEPS]
SECTION: Behavioral Task
1. Monkeys performed a programmed eye movement task in which they would first fixate on a central cue displayed on a monitor in front of the them for a fixed duration chosen from a range of 1.2 - 3 s (National Institute of Mental Health, National Institutes of Health, VCortex).  
SECTION: Behavioral Task
2. The central cue would then extinguish while a second target appeared on the screen's periphery, where the monkeys would have to saccade to and fixate for another 4 s before receiving a reward. 
SECTION: Behavioral Task
3. Whether the second target appeared to the left or right of the center cue was associated with whether successful completion of a trial would result in either a big or small reward. The second target's location/reward-size association would swap after 15 to 45 trials or 50 to 100 trials.
SECTION: Behavioral Task
4. The reward was a liquid food mixture (355 ml of Plus Nutrition Shake Vanilla, 355 ml of water, blended with two large bananas, and eight biscuits) delivered via a mouthpiece located near the animal's mouth at small and large reward volumes of 1.5 and 2.8 mL or 0.1 and 0.3 mL, respectively.
SECTION: Behavioral Task
5. Monkeys had a large reward probability of either 50% or 25% and an intertrial interval length of a fixed 7.5 s or a fixed value of 5 to 15 s per session. Subjects would perform between 500 and 1200 individual trials per recording session.
TITLE: Making CFM
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
TBD

[STEPS]
SECTION: Threading Carbon Fibre
1. Fill test tube with acetone (flammables cupboard) and add capillary tubes. 
SECTION: Threading Carbon Fibre
2. Stick test tube to light box with Blu Tack.
 

SECTION: Threading Carbon Fibre
3. Select a single carbon fibre using rubber tipped forceps.
SECTION: Threading Carbon Fibre
4. Thread carbon fibre down capillary tube. 
 

SECTION: Threading Carbon Fibre
5. Lift the tube out of the acetone and allow acetone to evaporate.
SECTION: Pulling Carbon Fibre
6. Place capillary tube into puller.

SECTION: Pulling Carbon Fibre
7. Tighten holder “finger-tight” and raise tube for 3 clicks and secure in place “finger tight”.
SECTION: Pulling Carbon Fibre
8. Settings: 
Magnet: off
Heat: ~6.7 

 

SECTION: Pulling Carbon Fibre
9. Check which end of the tube the carbon is in and carefully remove from puller.
SECTION: Pulling Carbon Fibre
10. Trim excess exposed fibre with fine scissors to stop waving extended fibre from stressing the glass seal.
SECTION: Cutting Carbon Fibre
11. Carefully place electrode between capillary tubes on cutting block.
SECTION: Cutting Carbon Fibre
12. Approximately line up seal with cut ridge.
 

SECTION: Cutting Carbon Fibre
13. Make a cut at ~ 300 µm from seal, by running scalpel down cut ridge, mark this as 0 on the graticule.
SECTION: Cutting Carbon Fibre
14. Move electrode so there is 50-100 µm exposed carbon fibre and run scalpel down cut ridge. 
 

SECTION: Cutting Carbon Fibre
15. Carefully remove electrode from cutting block.
SECTION: Wiring Electrode
16. Cut wire to slightly longer than your capillary tube.
SECTION: Wiring Electrode
17. Strip off ~1cm of the plastic coating from each end.
SECTION: Wiring Electrode
18. On one end trim exposed wires so only 1 is sticking out.
SECTION: Wiring Electrode
19. Dip this end in the conductive silver paint and gently thread down the capillary so the paint makes contact with the carbon fibre.
SECTION: Wiring Electrode
20. Glue exposed end of wire in place by dripping glue down capillary tube, exposed wire can be connected to voltammetry head-stage using croc-clip. 
SECTION: Wiring Electrode
21. Allow to set overnight before use.
TITLE: GUS staining
AUTHORS: lilyli

[DESCRIPTION]
GUS staining

[STEPS]
1. Fix with 90% 90 % volume acetone for 20 min 

2. Add 1 mLGus washing solution to wash off the acetone and wash it twice

3. Add Gus staining solution and vacuum on ice for 15-20 min
4. 37 ℃ overnight
5. Remove the dyeing solution, add 1ml of 70% ethanol
6. Change the ethanol several times
7.
TITLE: Direct-Blot™ Western Blotting Protocol
AUTHORS: Sam Li

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

[STEPS]
?. [Sample Preparation:]
Place cells in a microcentrifuge tube and centrifuge to collect the cell pellet.
?. [Sample Preparation:]
Lyse the cell pellet with 100µl of lysis buffer on ice for 30 min (For 1 X 106 cells, lyse with 100µl of lysis buffer).
?. [Sample Preparation:]
Centrifuge at 14,000 rpm (16,000xg) for 10 minutes at 4°C.
?. [Sample Preparation:]
Transfer the supernatant to a new tube and discard the pellet. Remove 20µl of supernatant and mix with 20 µl of 2x sample buffer.
?. [Sample Preparation:]
Boil for 5 min. Cool at room temperature for 5 minutes. Microcentrifuge for 5 minutes.
?. [Sample Preparation:]
Load up to 40µl of sample to each well of a 1.5mm thick gel.Note: Guidelines for choosing gel percentages are based on protein size to be detected: 4-5% gel, >200 kD; 7.5% gel, 120-200 kD; 8-10% gel, 40-120 kD; 13% gel, 15-40 kD; 15% gel,
?. [Sample Preparation:]
Set gel running conditions according to the manufacturer’s instructions. Transfer the proteins to a nitrocellulose or PVDF membrane with variable power settings according to the manufacturer’s instructions.
?. [Membrane Blocking:]
Remove the blotted membrane from the transfer apparatus and immediately place in blocking buffer consisting of 5% nonfat dry milk/TBS-T.Note: If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk.
?. [Membrane Blocking:]
Incubate the blot for 1 hour at room temperature, or overnight at 4°C with agitation.
?. [Antibody Incubation:]
Dilute the Direct-Blot™ antibody to the recommended concentration/dilution in 5% nonfat dry milk/TBS-T (usually at a 1:1000-1:2000 dilution). Place the membrane in the Direct-Blot™ antibody solution and incubate for 2 hours at room temperature, or overnight at 4°C with agitation.Note: If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk.
?. [Antibody Incubation:]
Wash three times for 5 minutes each with Wash Buffer (TBS containing 0.1% Tween-20)
?. [Protein Detection:]
Incubate membrane (protein side up) with 10ml of ECL (enhanced chemiluminescence substrate) for 1-2 minutes. The final volume required is 0.125ml/cm2.
?. [Protein Detection:]
Drain off the excess detection reagent, wrap up the blots, and gently smooth out any air bubbles.
?. [Protein Detection:]
Place the wrapped blots, protein side up, in an X-ray film cassette and expose to x-ray film. Exposures can vary from 5 seconds to 60 minutes.
TITLE: Serial Sectioning of Mouse Brain
AUTHORS: Asta Zane, Nicole J Corbin-Stein, Gabrielle Childers, Jhodi Webster, Vickie Yang, Woong-Jai Won, Rajesh Gupta, Ashley Harms

[DESCRIPTION]
This protocol highlights sectioning 40 micron mouse brain tissue to be used for histology experiments. 

[GUIDELINES]
Make sure to punch the hole in the non-injected side of the brain. That will allow to quantify the staining much easier later. If injected bilateral, then do not punch the hole. The hole location will be different for different parts of the brain: for striatum it will be in the cortex, and for SNpC in the middle.

[STEPS]
SECTION: PROCEDURE
1. Label 24 or 12 well plate with appropriate identifiers (date, project, region to be sectioned etc.) and fill each well ⅔ full with anti-freeze solution (50% glycerol in PBS).
SECTION: PROCEDURE
2. Freeze microtome stage on the dry ice.
SECTION: PROCEDURE
3. Crush dry ice for the sprinkling the stage later
SECTION: PROCEDURE
4. When ready place the stage into place, level it. Put the dry ice into it to keep it cold while working. 
SECTION: PROCEDURE
5. Put the microtome blade into the place, keep it covered not to get injured.
SECTION: PROCEDURE
6. Place few drops of sucrose on the cold stage and put your brain on it. The brains bottom will be hindbrain, and the top of the brain will be the forebrain. The brain lateral side will be facing you, and dorsal away from you. Put few more drops of sucrose to support the brain. Sprinkle crushed dry ice around the brain and cover the whole stage with foul for 2-3 min, to fully freeze.
SECTION: PROCEDURE
7. Prior to sectioning, adjust the thickness setting, all sections will be 40 microns thick, and trimming has to be 80 microns. This knob is found on the side of the microtome.
SECTION: PROCEDURE
8. Slowly move the blade towards you, slicing a section of the brain. To prevent the sections from folding and breaking, slice slowly and in a single continuous motion. 
SECTION: PROCEDURE
9. Gently scoop the brain section away from the blade, using a thin painting brush. Slowly place the brush into antifreeze solution. Gently move the brush to allow the section to unfold in the liquid. Make sure the sections go into appropriate wells.
SECTION: PROCEDURE
10. Continue slicing until you have sliced the entirety of the brain.
SECTION: PROCEDURE
11. Secure the well plate with tape. Store brain sections in antifreeze solution at -20C.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Reagent List:</strong><br />
<ul>
<li>Cell Staining Buffer (BioLegend Cat. No. 420201)</li>
<li>Red Cell Lysis Buffer (BioLegend Cat. No. 420301)</li>
<li>7-AAD Viability Staining Solution (BioLegend Cat. No. 420403)</li>
<li>TruStain FcX™ (anti-CD16/32, BioLegend Cat. No. 101319)</li>
<li>Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301)</li>
</ul>
<br /><strong>References:<br /></strong>Current Protocols in Cytometry (John Wiley &amp; Sons, New York), Unit 6 Phenotypic Analysis.<strong><br /></strong>

[STEPS]
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