eamag/protocols-with-steps huggingface dataset:
TITLE: Fecal Output Protocol
AUTHORS: Adam Hamilton, Ian N Krout, Tim Sampson

[DESCRIPTION]
This assay is used to quantify the number of fecal pellets produced over a short period of time, which serves as a measure of colonic motility. Mice are placed into individual clear plastic beakers and the number of fecal pellets in the container are counted every 5 minutes over a 30 minute period. 

[STEPS]
SECTION: Prior to Assay
1. Prepare clean 1 liter (~12cm x 25cm) translucent beakers- sterilize if necessary, with
aluminum foil covers. These should be autoclaved before use if collection of fecal pellets for microbiome analysis is required.
SECTION: Day of Set-up
2. Bring mice to testing room for at least 60 min prior to assay, to acclimate (if testing mice in the same room they are housed no adjustment period is necessary).

SECTION: Day of Assay
3. Separate 1L beakers and cut/rip foil into lids to cover each beaker individually. 
SECTION: Day of Assay
4. Place each mouse individually into a beaker, cover with foil, and start timer. Generally, 8-10 animals can be
handled at once before start times begin to overlap.
SECTION: Day of Assay
5. Do not leave animals unattended while in beakers- they will attempt to jump out. 
SECTION: Day of Assay
6. In 5 min intervals, gently lift each cylinder, and count the number of fecal pellets present on the beaker floor and walls. Record the number of pellets for a minimum of 15 min, up to30 min. 

SECTION: Day of Assay
7. Because mice are coprophagic, some pellets may disappear (be eaten) over this experiment. To be consistent, record only the cumulative number of pellets observed. Pellets produced cannot
“decrease” so the value should either stay level or increase in each 5 minute bin over the course of this assay. 
SECTION: Day of Assay
8. Return animals to home cage, collect fecal pellets if needed. 
SECTION: Analysis
9. Comparisons should be made between genotypes and treatment groups as to the amount of pellets produced in 30 min. There should also be comparisons of the amount of pellets produced at each 5 min interval.
TITLE: Fungal Extraction from Beetle Galleries
AUTHORS: You Li, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to extract fungi from beetle galleries. </div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Crystallization of MERS-CoV Mpro
AUTHORS: blake.h.balcomb, Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson

[DESCRIPTION]
 The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus. 

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
32 µL17 mg/mL  
3.36 mL 
14.4 µL   seeds, dilution 1:1000

SECTION: Crystallization experiment
5. Dispense 35 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL17 mg/mL    to each lens using the SPT mosquito.
Dispense  20 nL to each lens using the SPT mosquito.
Dispense  130 nL to each lens using the SPT mosquito.

Drop ratio: 15:13:2 ratio (150 nl   : 150 nl reservoir solution: 20 nl seeds)
Final drop volume: 300 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.2 Molarity (M)  Sodium malonate dibasic monohydrate
20% w/v PEG 3350

Stock solutions used:
JCSG+ condition 2-30

 

SECTION: Crystallization experiment
7. Crystal form after ~24 h.
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 1000 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Melting and alloying operations were performed in a low carbon steel crucible in an inert ultra-high purity argon environment, preventing the oxidation reactions of molten Mg. High purity Mg (99.97%) was heated to 710°C for 10 minutes until the slug was completely melted. Each alloying element was added separately and kept for 10 minutes before additional alloy was added to ensure complete dissolution of all alloying elements. Automated stirring was applied for 15 minutes to ensure a homogeneous distribution of elements throughout the melt. The melt was then raised to 730 °C, held for 15 minutes and then poured into a steel tube mold. A solution heat treat was carried out on the as-cast alloy at 510 °C for 10 h before it was extruded. The extrusion was conducted with an extrusion ratio 10, extrusion temperature 400 ºC and ramp speed 1 mm/s.</p>

[STEPS]
TITLE: Experimental protocol for data collection of Datura spp.
AUTHORS: Eunice   Kariñho-Betancourt

[DESCRIPTION]
Experimental design employed to obtain biological materials for RNA seq analysis. We collected leaf tissue to examine gene family evolution of four Datura species. 

[STEPS]
SECTION: Germination
1. Plants were started from seed between March-April, growing under 16:8, L:D cycle with 25:20°C (L:D) in the glasshouse of the Institute of Ecology, National Autonomous University of Mexico. Experimental plants were obtained by sowing seeds of maternal families (natural progenies). Once the true leaves appeared and seedlings reached 3-5 cm long, they were planted in single 150 mL pots (Figure 1), in sterilized soil, and watered ad libitum.



  

SECTION: Leaves collection
2. Nine plants (of different maternal families) were collected at two developmental stages. To capture developmental variation 10 fully expanded leaves from each plant were harvested either at the vegetative or reproductive stage as follows:
When plants reached at least 15 cm and/or started dichotomous branching (~40 days after germination), three juvenile plants were defoliated, whereas the six other plants remained undamaged until flowering.

Since leaf age of vegetative plants at the time of first harvesting differed from mature plants at the final harvesting, we controlled for leaf age differences at the two collection episodes by marking the new leaves produced after the first harvesting, in both treatment-allocated plants so that they could be compared.
When the first flower emerged, at the reproductive stage (~ 60 days after germination), leaves of the remaining six plants were harvested. All leaves were flash-frozen after collection and stored at -80°C until RNA extraction.
TITLE: Isolation of total DNA from Synechocystis sp. PCC 6803
AUTHORS: Anna Behle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol can be used for extraction of total genomic DNA from </span><span style = "font-style:italic;">Synechocystis</span><span> sp. PCC 6803.</span></div></div>

[STEPS]
?. [Culturing]
Grow 50 mL Synechocystis culture to the end of the logarithmic phase (OD750 ≈ 1.0).Centrifuge at 4800 rpm and 4°C for 7 minutes. Remove supernatant.
?. [Wash steps:]
Resuspend cells in 10 mL TE-buffer, centrifuge at 4800 rpm and 4°C for 7 minutes. Remove supernatant.
?. Repeat washing step. Resuspend pellet in 1 mL TE-buffer. Flash freeze in liquid nitrogen or dry ice.
?. [Buffers required:]
TE-Puffer : 10 mM Tris/HCl ; 1 mM EDTA ; pH 8.0TE+S-Puffer : 25% (w/v) Sucrose; 50 mM Tris/HCl ; 1 mM EDTA, pH 8.0
?. [Cell lysis]
Add 5 mg · mL-1 lysozyme  (optional: + 100 mM EDTA).Incubate 1 hour at 37˚C.
?. Add 14,4 µL Proteinase K (20 mg/mL stock) + 100 µL 20 % SDS.Incubate 1 hour at 60 ˚C. (Alternatively, incubation can be carried out at 37 ˚C for 16 hours).
?. [DNA extraction:]
Add 1 volume of Phenol/Chloroform (1:1 v/v). Mix well.Centrifuge for 10 min at 13000 rpm and 4° C.
?. Transfer upper, aqueous phase to a fresh tube.Add 1 volume of Phenol/Chloroform (1:1 v/v). Mix well.Centrifuge for 10 min at 13000 rpm and 4° C.
?. Transfer upper, aqueous phase to a fresh tube.Add 1 volume of Chloroform in order to remove residual phenol. Mix well.Centrifuge for 10 minutes at 13000 rpm and 4° C.
?. [DNA precipitation]
Add 0.7 volumes of isopropanol to the sample. Incubate for 5 minutes at RT.
?. Centrifuge for 30 minutes at 13000 rpm and 4° C.
?. Wash pellet with 1 mL 70 % EtOH.Centrifuge for 10 minutes at 13000 rpm and 4˚C.
?. Air-dry pellet for 1 hour.
?. [RNase treatment:]
Resuspend DNA pellet in 30-100 µL TE-buffer.Add 2 µL RNase AIncubate 30 minutes at 37˚C.
?. [RNase treatment:]
Heat inactivate RNase A by incubating for 10 minutes at 72 ˚C.
TITLE: Contractile response to chemogenetic activation or inhibition of cholinergic or nitrergic myenteric neurons of the mouse colon
AUTHORS: Thomas Gould, Dante Heredia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for measuring colonic migrating motor complexes in response to chemogenetic activation or inhibition of cholinergic or nitrergic  myenteric neurons of the mouse colon</div></div>

[STEPS]
?. A ventral midline incision is made and the whole colon is carefully excised into a Sylgard-lined dissection dish containing oxygenated Krebs-ringer solution.
?. The colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon.
?. An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle.
?. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening.
?. Suture silk is used to connect three force transducers (model TST125C; Biopac Systems, Santa Barbara, CA) to the proximal, transverse and distal segments of the colon.
?. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems).
?. After spontaneous colonic migrating motor complexes (CMMCs) are detected, 10 mM clozapine-n-oxide is perfused into the bath.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is used to clarify the process of total DNA extration for our R. crenulata genome.</p>

[STEPS]
?. 
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?. 
?. 
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?. 
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?.
TITLE: iNDI PiggyBac-TO-hNGN2 transfection protocol Version 1
AUTHORS: Mark  Cookson, michael.ward  , Erika  Lara Flores

[DESCRIPTION]
PiggyBac Method for hNGN2 transfection
Transfection protocol
Use of CEPT: Nature Methods 18, 528-541, 2021

[STEPS]
SECTION: Transfection protocol
1. Observe KOLF2.1 iPSCs under a phase contrast microscope to assess confluency and presence of cells debris. Dish should be dissociated at ~70% to 90% confluency.

Coat a well of 6 well plate to be used for transfection with 1 mL of Matrigel solution, tilting to ensure coverage of entire surface area. Place in 37 °C incubator for 30 min .



SECTION: Transfection protocol
2. Prepare E8 medium supplemented with CEPT and place in at Room temperature to warm during dissociation.

 

SECTION: Transfection protocol
3. Aspirate culture medium from well/plate that should be dissociated and wash with PBS 1X.

SECTION: Transfection protocol
4. Aspirate PBS and add half of culture volume of Accutase. 
SECTION: Transfection protocol
5. Transfer to 37 °C incubator for 10 min .

 

SECTION: Transfection protocol
6. Meanwhile aspirate Matrigel from well and add 2 mL culture medium E8 supplemented with CEPT.

SECTION: Transfection protocol
7. When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to break the colonies.

SECTION: Transfection protocol
8. Quench the Accutase adding half of the culture volume of PBS.

SECTION: Transfection protocol
9. Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.

SECTION: Transfection protocol
10. Centrifuge 5 min at 200 - 300 x g at Room temperature .
SECTION: Transfection protocol
11. Aspirate supernatant and resuspend the cell pellet in culture medium E8 supplemented with CEPT.
SECTION: Transfection protocol
12. Count cells and plate 1 - 1.5 x 106  cells into a Matrigel-coated well. Gently swirl plate to evenly distribute cells.

SECTION: Transfection protocol
13. Return plate to 37 °C incubator.
SECTION: Transfection protocol
14. After ~ 60 min to 120 min of plating cells , warm OptiMEM at Room temperature .
 




SECTION: Transfection protocol
15. Prepare one tube with:
200 µL of OptiMEM
Total of 3 µg of DNA mix in a 1:3 (Transposase:DNA) ratio
 

5 µL to 7.5 µL  of Lipofectamine Stem 
 


SECTION: Transfection protocol
16. Incubate transfection mix for 15 min to 30 min at Room temperature .

 

SECTION: Transfection protocol
17. Add transfection mix from step 15 to cells drop wise and immediately swirl the plate to evenly distribute the mix to cells.
SECTION: Transfection protocol
18. Return plate to 37 °C incubator.
SECTION: Transfection protocol
19. Next day, check for nuclear BFP positive signal (for PB-TO-hNGN2-BFP plasmid). If a positive transfection occurred and the well is confluent, re-plate the cells and puromycin select 24-48 h after transfection (it all depends on the health and transfection efficiency of the cells) with 8 µg/mL  for 2 to 14 days.

 


SECTION: Transfection protocol
20. Change media accordingly and expand cells when they reach 70-90% confluency.
TITLE: Top agar: 0.7% w/v LB(Miller) agar
AUTHORS: HANNAH ZHU

[DESCRIPTION]
how to make Top agar for plaque assay experiment

[STEPS]
SECTION: Top agar: 0.7% w/v LB(Miller) agar
1. Dissolve following components in 200 mL MilliQ water

 


SECTION: Top agar: 0.7% w/v LB(Miller) agar
2. Autoclave at 121 °C for 15 min

SECTION: Top agar: 0.7% w/v LB(Miller) agar
3. Store in oven (> 50 °C)
TITLE: Mitophagy induction using Oligomycin/Antimycin A
AUTHORS: Louise Uoselis

[DESCRIPTION]
Mitophagy induction in HeLa cells using Oligomycin/Antimycin A.

[STEPS]
SECTION: Day 1
1. Seed cells, aiming for a confluency of 80-90% at the time of treatment the next day. 
SECTION: Day 2
2. Feed cells for 60 min in an appropriate volume of standard growth media. 

SECTION: Day 2
3. To start the treatment, replace the media in each well with standard growth media that contains 10 micromolar (µM) Oligomycin, 4 micromolar (µM) Antimycin A, and 10 micromolar (µM) QVD. 

SECTION: Day 2
4. Harvest the samples after the desired treatment times.
TITLE: Transformation of Arabidopsis Thaliana Protocol
AUTHORS: creative-biogene

[DESCRIPTION]
In this experiment, the flower immersion method was used to transfer the target gene into Arabidopsis thaliana using Agrobacterium mediated.

[STEPS]
SECTION: Experiment Summary
1. In this experiment, the flower immersion method was used to transfer the target gene into Arabidopsis thaliana using Agrobacterium mediated.
SECTION: Main Reagents
2. YEB liquid medium, LB medium, 0.1 M CaCl2, 0.05 M MgSO4, flower immersion buffer (0.5XMS, 5% sucrose, 0. 03% Silwet L-77), Rif, Kan.
SECTION: Main Equipment
3. Shaker, centrifuge, culture bowl, greenhouse, trays, plastic film.
SECTION: Experimental Materials
5. Constructed expression vector, Arabidopsis thaliana.
SECTION: Experimental Steps
4. 1. Preparation and transformation ofAgrobacterium tumefaciensreceptor cells
(1) Pick a single colony of GV1301 in 10 ml of YEB or LB liquid medium and incubate with shaking at 28°C until late logarithmic phase.
(2) Add 0.5 ml of bacterial solution to 50 ml of fresh YEB or LB liquid medium and incubate at 28°C with shaking until OD600=0.5.
(3) Transfer to 50 ml centrifuge tube and ice bath for 20 min.
(4) Transfer to a 50 ml centrifuge tube and ice bath for 20 min.
(5) Centrifuge at 4000 rpm for 10 min at 4°C and collect the organisms.
(6) Resuspend the precipitate with 8 ml of pre-cooled 0.1 M CaCl2and 0.05 M MgSO4.
(7) Centrifuge at 4000 rpm for 10 min at 4°C and collect the bacteriophage.
(8) Take 200 ul of receptor cells, add 0.5 ug of plasmid and mix gently.
(9) Freeze in liquid nitrogen for 50 s, place on ice for 40 min, and heat-excite at 42°C for 90 s.
(10) Add 800 ul of LB medium and incubate for 1 hour at 28°C.
(11) Apply the appropriate amount of cells on the screening medium and blow dry the surface liquid on the ultra-clean table.
(12) Incubate at 28°C for two days.
2. Transformation ofArabidopsis thalianaby flower immersion
(1) Inoculate Agrobacterium clones containing expression vectors in 5 ml YEB medium (containing 100 ug/ml Rif, 100 ug/ml Kan) at 28°C, 200 rpm and incubate with shaking overnight.
(2) Transfer to 200 ml YEB medium at a ratio of 1:50 and incubate at 28°C, 200 rpm for 5 hours; centrifuge at 5000 X g for 15 min and collect the bacteriophage; resuspend in floral immersion buffer (0.5XMS, 5% sucrose, 0. 03% Silwet L-77) and adjust OD600to 0.8.
(3) Place the Arabidopsis culture bowl upside down on a suitable size container containing flower soaking buffer, soak for 3-5 min, remove the culture bowl upside down in a tray, cover the tray with plastic film, remove the film after 24 hours, and continue the culture in the greenhouse.
TITLE: Image analysis of plasma membrane contacts
AUTHORS: Chase Amos, Pietro De Camilli

[DESCRIPTION]
This protocol details the image analysis of overexpressed VPS13A^Halo at plasma membrane contact sites in K562 cells.

[STEPS]
SECTION: Image analysis of plasma membrane contacts
1. Using the image analysis program FIJI, split the channels of the image containing the mitochondria (TMRE stain or overexpressed mito-BFP) and overexpressed VPS13A^Halo.
SECTION: Image analysis of plasma membrane contacts
2. Threshold the mitochondria channel to include the mitochondria. Convert to a 32-bit image and convert the threshold to binary, setting values within the mitochondria to be NaN (not a number). This step generates a negative mitochondrial mask.
 

SECTION: Image analysis of plasma membrane contacts
3. Multiply the VPS13A^Halo channel and the negative mitochondrial mask into a 32-bit result image. This generates a mitochondria-subtracted VPS13A^Halo image.
SECTION: Image analysis of plasma membrane contacts
4. Using the mitochondria-subtracted VPS13A^Halo image, apply a threshold to include signal present at the plasma membrane. Convert to a 32-bit image and convert threshold to binary, setting values outside of the VPS13A signal to be NaN. 

The resulting image includes plasma membrane and excludes signal originating from the mitochondria or background.
SECTION: Image analysis of plasma membrane contacts
5. Trace the cell edge using the selection brush tool with a pixel size of 1. Apply the ROI to the image generated in step 4 and measure the "% area" (selected as "Area fraction" in the "Set measurement" window) of the cell edge length. The recorded value corresponds to the percent of plasma membrane length that contains VPS13A^Halo signal.
TITLE: Exiting the Lab (Covid-19 Shutdown Re-Entry, Srivastava Lab)
AUTHORS: Srivastava Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Disclaimer: </span><span>The SOP presented here has been designed by the Srivastava Lab at Harvard University and is being shared outside of Harvard for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
?. If it is your “high touch” day, wipe all handles (incubator, freezer, door, etc) with 70% ethanol
?. Wipe bench piettes, etc with 70% ethanol
?. Wipe phone/other objects in workspace (laptops, etc) with 70% ethanol
?. Wash hands at the sink
?. Turn on water
?. Wash hands
?. Dry hands with paper towel
?. Turn off faucet with paper towel