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

[DESCRIPTION]
This cloning protocol refers to the Marburg Collection

[BEFORE_START]
Before start, the resistance and ori parts have to be digested with BsaI and purified!

[GUIDELINES]
This cloning protocol refers to the Marburg Collection

[STEPS]
SECTION: Predigestion
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SECTION: Predigestion
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SECTION: Reaction Setup on ice:
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SECTION: Reaction Setup on ice:
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SECTION: Reaction Setup on ice:
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SECTION: Reaction Setup on ice:
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SECTION: Reaction Setup on ice:
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SECTION: Thermocycling conditions
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SECTION: Thermocycling conditions
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SECTION: Thermocycling conditions
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SECTION: Thermocycling conditions
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TITLE: Immobilizing cells for live imaging
AUTHORS: Tara Essock-Burns, David Matus

[DESCRIPTION]
This protocol covers the sample preparation for high-resolution confocal microscopy to visualize algal cells and their organelles (chloroplasts and mitochondria) by immobilizing them in agarose. The sample preparation includes preparing low-gelling agarose, staining the cells, and preparing the slides with live, but immobilized, algal cells. 

[STEPS]
SECTION: Prepare low-melt agarose in appropriate medium
3. To make 1.25% low-melt agarose in TAP medium (which we use for Chlamydomonas cells), measure 3.75 mg of low-melt agarose and dissolve into 3 mL of TAP medium in a glass culture tube, then place it on a heat block set to 100 °C until the agarose dissolves. 
 

SECTION: Prepare low-melt agarose in appropriate medium
4. Once dissolved, move the tubes to a heat block set at 45 °C and let the mixture acclimate for at least 20 min.
SECTION: Staining the cells
6. Pellet the cells by microcentrifugation 2000 × g for 2 min. Remove the supernatant and discard it.
SECTION: Prepare the cells in appropriate medium
1. Scoop half of a sterile loop of cells (here we used Chlamydomonas reinhardtii and C. smithii) from a lawn plate and vigorously agitate the loop along the inside of a 1.5 mL microcentrifuge tube containing 500 µL of the medium for the experiment (TAP medium or ddH2O). 
SECTION: Staining the cells
5. Prepare a working stock solution of vital dyes in an appropriate medium. For PKmito ORANGE, we used 1:500 in TAP medium. For staining two species of Chlamydomonas, we prepared 1 mL of dye to split (500 µl for each tube).
SECTION: Prepare the cells in appropriate medium
2. Leave the tubes with cells in a tube rack on the bench overnight with a light source illuminating them under a 12:12 hr light:dark cycle.
SECTION: Staining the cells
7. Add 500 µL of the working stock of dye to each tube and mix by gently pipetting up and down, resuspending the pellet.
SECTION: Staining the cells
8. Transfer the cell/dye mixture to an opaque microcentrifuge tube (or wrap the tube with foil) to protect it from light and secure the cap with Parafilm. 
SECTION: Staining the cells
9. Place the tubes on a rotator to incubate with the dye for 45 min (at room temperature).
 

SECTION: Staining the cells
10. After incubation, pellet and wash the cells twice with fresh TAP medium.
SECTION: Mounting the cells
11. Draw a small wax circle (using a wax pencil) on a #1.5 glass coverslip (18 × 18 mm) for each sample (two separate coverslips for two tubes).
SECTION: Mounting the cells
12. After removing the supernatant following the second wash, resuspend the pellet in 25 µL of the warmed (45 °C) agarose mixture.
SECTION: Mounting the cells
13. Pipette 1 µL of the agarose/cell mixture within the wax circle on the coverslip.
SECTION: Mounting the cells
14. Using forceps, carefully flip the coverslip onto a glass slide and seal the edges with VALAP.
TITLE: Micro-CT scanning and fiber localization
AUTHORS: Mai-Anh Vu, mwhowe

[DESCRIPTION]
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the micro-CT scanning and fiber localization steps. Please contact us (mwhowe@bu.edu) if you are interested in using this technique. 

[STEPS]
SECTION: Perfusion and dissection
1. Mice were injected intraperitoneally with 400-500 mg/kg Euthasol (Covetrus Euthanasia III), and then perfused transcardially with 20mL 1% phosphate buffered saline (PBS, Fisher), followed by 20mL 4% paraformaldehyde in 1% PBS.
SECTION: Perfusion and dissection
2. After perfusion and decapitation, the lower jaw and front of the skull were removed in order to allow diffusion of solution into the brain while still keeping the implant intact.
SECTION: Preparation for CT scanning
3. The brain was soaked in the 4% paraformaldehyde solution for 24h, rinsed three times with 1% PBS, and then transferred to a diluted Lugol’s solution, to provide tissue contrast for computerized tomography (CT) scanning (Metscher, 2009).
SECTION: Preparation for CT scanning
3.1. The Lugol solution was prepared by diluting 10mL 100% Lugol’s Solution (Carolina, 10% potassium iodide, 5% iodine) with 30mL deionized water, a dilution chosen to be approximately isotonic to biological tissues61.
SECTION: Preparation for CT scanning
3.2. Note on soak time: Initially, samples were soaked in this diluted Lugol’s solution in a foil-wrapped 50mL conical centrifuge tube on an orbital shaker plate for 10-14 days. We have more recently found that using 4 oz specimen cups instead of the 50mL conical centrifuge tubes enables better diffusion of the Lugol’s solution, and adequate contrast can be achieved in three to four days.
SECTION: Preparation for CT scanning
4. After soaking, the skulls were rinsed three times with 1% PBS, and secured in a modified centrifuge tube.
SECTION: Micro-CT scanning
5. The implanted skulls were imaged in a micro-CT scanner (Zeiss Xradia Versa 520, a core instrument of the Boston University Micro-CT and X-ray Microscopy Imaging Facility) with the following parameters: 140kV, 10W, HE1 filter, 0.4X objective, 2s exposure time, 1001 projections, 12-micron voxel size.
SECTION: CT registration and fiber localization
6. The CT was then registered to the Allen Mouse Brain Common Coordinate Framework 3D 10-micron reference atlas (Wang et al., 2020) to bring individual mice into a common coordinate system, and then fibers were identified and mapped from the recording tip up to the grid. This process was carried out using a combination of FIJI (https://imagej.net/software/fiji/) and MATLAB (Mathworks, version 2020b) using a combination of existing MATLAB functions and custom
lab-written functions and GUIs. This pipeline is now publicly available at https://github.com/HoweLab/MultifiberLocalization.
TITLE: Optical density measurement for Bacterial solution (Worm Food)
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Before the OP50 solution is aliquoted into small falcon tubes, it is always better to check the OD by using a spectrophotometer, for documentation purposes.</div></div>

[STEPS]
?. Pre-measurement Clean the disposable cuvettes with ethanol and lint free paper Aliquot small amount of LB broth into a falcon tube (For Blank) Fill about 1ml of the LB broth into a clean cuvette Mix/Vortex the tube containing the stock bacteria to achieve a homogenized solution Take three aliquots from the same tube in three different cuvettes
?. Actual measurement Switch on the spectrophotometer Press the bottom left button to select the OD program Increase the wavelength to 600nm Put the blank cuvette first and press the button showing blank Put the sample cuvette 1 and press the button showing sample Take a note of the OD reading Place the second sample and repeat After taking readings for all three samples take the average Record the OD as OD600 = 1.35
?. Post measurement Discard the cuvettes Close the lid to the spectrophotometer Switch it OFF
TITLE: Chimeric Protein-LA and Protein LAG sandwich ELISA 
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein LA (SpLA) and protein-LAG (PLAG) with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein LA (SpLA) and protein-LAG (PLAG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpLA in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled PLAG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.30.
TITLE: Arthrospira platensis Growth Media Stock Solution Preparation
AUTHORS: Victor vmr Rodriguez

[DESCRIPTION]
The purpose of this protocol is to prepare a growth media for
spirulina (Arthrospira platensis). Arthrospira
platensis is a cyanobacterium species usually found in alkali
environments rich with carbonate and bicarbonate concentrations. Arthrospira
platensis cultures thrive in media with a pH between 9 and 10, with high
bicarbonate concentrations, inorganic salts, and trace Nutrients. 

[BEFORE_START]
Ensure you are working in a sterile aseptic environment 

[STEPS]
SECTION: Materials Needed
1.1. 500 ml media bottle
SECTION: Materials Needed
1.2. magnetic stir bar
SECTION: Materials Needed
1.3. Weight boat
SECTION: Equipment Needed
2.1. UV Sanitizer
SECTION: Materials Needed
1.4. P1000 pipettor
SECTION: Materials Needed
1.5. P1000 pipette tips
SECTION: Materials Needed
1.8. Roll of labeling tape
SECTION: Materials Needed
1.7. Magnetic stir bar remover magnet
SECTION: Materials Needed
1.6. Spatula
SECTION: Equipment Needed
2.2. Magnetic Stirrer 


SECTION: Reagents Needed
3.1.  

SECTION: Reagents Needed
3.2.   

SECTION: Materials Needed
1. Gather Materials
SECTION: Equipment Needed
2. Gather Equipment
SECTION: Reagents Needed
3. Gather Reagents
SECTION: Methods
4. Prepare Media
SECTION: Methods
4.1. Fill the500 mL media bottle with500 mLof  

SECTION: Methods
4.2. Add the magnetic stir bar to the media bottle and turn on the mixer to medium
SECTION: Methods
4.3. Add 7.5 g of   to the media bottle

SECTION: Methods
4.4. Add 1.0 mL of   to the media bottle

SECTION: Methods
4.5. Allow the media bottle to stir until all the salts have dissolved (You may need to move the media bottle around so that the stir bar agitates all the settled nutrient salts)
SECTION: Methods
4.6. Using a magnet, remove the magnetic stir bar from the media bottle
SECTION: Methods
4.7. Place the media bottle in UV sanitizer for 10 minutes uncapped, with cap facing up
SECTION: Methods
4.8. First place cap on media bottle, Remove the media bottle from the UV sanitizer
SECTION: Methods
4.9. Label media bottle “Spirulina Culture Media” and date
SECTION: Reagents Needed
3.3.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol for making and running a 12% SDS-PAGE Western blot. Protocol starts from pouring gels all the way to reading the membrane. </p>

[BEFORE_START]
<p>Run a BCA assay before starting a Western Blot</p>
<p>Make 10% AP fresh each time you pour gels. Mix 0.1 grams of AP with 100 microliters of waters.</p>
<p> </p>

[GUIDELINES]
<p>Not all Western blots are going to work out. Prepare for failures.  </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for sampling people in order to understand patterns of mobility and activity-space segregation at the scale of city-days. The goal is to obtain a representative sample of the space-time paths that exist in a given city. Sampling points and times are choosen and then used to recruit participants. If participants consent to be part of the study, they (1) respond anonymously to a survey about their activity-spaces, and/or (2) use a mobile phone application to track themselves during a 1-week period and share their anonymized trajectory information with the research team. This protocol has been developed thanks to funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 657956. </p>

[GUIDELINES]
<p>Ethics guidelines:</p>
<ul>
<li>Volunteers should be accepted into the study only if they provide informed consent</li>
<li>Volunteers should be accepted into the study only if they are adults and are not incarcerated, elderly, pregnant, mentally disabled or otherwise in a position that would put them at particular risk or add additional bases of vulnerability.</li>
<li>The sampling method should be designed to produce gender balance in the sample.</li>
<li>No personally-identifying information should be collected from participants.</li>
<li>All location information collected by the survey and mobile phone tracking application is placed into a predefined grid of 4 hectare cells. Only the cell identifier, not the exact location, is recorded. </li>
<li>All time information collected by the survey and mobile phone tracking application is rounded down to the nearest 15 minute interval. No exact times are recorded.</li>
</ul>

[STEPS]
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TITLE: Screening Edited iPSC Clones
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Screening Edited iPSC Clones</span></div><div class = "text-block"><span style = "font-weight:bold;">Expanding and gDNA Extraction from iPSC Clones</span></div><div class = "text-block">Note: It takes approximately 1 week for iPSC picked into 96 well plates to be sufficiently confluent for freezing and screening. For screening purposes, a fraction of the cells picked into one well of a 96 well plate will be saved for DNA Extraction and the remaining will be kept in culture or frozen down. </div></div>

[STEPS]
TITLE: Elisa_Protocols_Cytokines
AUTHORS: Marcia Guimaraes da Silva, Jossimara Polettini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunoenzymatic test (enzyme-linked immunosorbent assay) for cytokines: Interleukin (IL)-1, IL-6, IL-8 and Tumor necrosis factor (TNF)-a</div></div>

[STEPS]
TITLE: Polymerase Chain Reaction for the Identification of a Plasmid
AUTHORS: SGD

[DESCRIPTION]
Protocol for the identification of a plasmid by polymerase chain reaction followed by gel electrophoresis

[STEPS]
SECTION: Making the PCR mix
1. Add 1 ng of the target DNA, 
5 µL of each primer, 
25 µL of 2X PCR master mix, 
and sufficient sterile water to make the volume up to 50 µL.

SECTION: Thermocycling
2. Thermocycle on the following settings:

 

SECTION: Making the Gel
4. Add 100 mL 1× TAE buffer and swirl gently to mix. Put an inverted small conical flask in the top of the flask.

SECTION: Making the Gel
5. Place in a microwave and heat for 1 minute, then gently swirl the contents. 
 

SECTION: Making the Gel
6. Continue microwaving in 30-second blasts followed by gentle swirling until the agarose starts to boil. 

 


SECTION: Making the Gel
7. Continue microwaving in 15-second blasts until all the agarose has dissolved - you should not be able to see any tiny pieces floating in the gel suspension.
SECTION: Making the Gel
8. Place the flask in a 50 °C water bath to cool. It is important to make sure the agarose has cooled to 50 °C before proceeding to the next stage.

SECTION: Making the Gel
9. Whilst you are waiting for the gel to cool, prepare your gel tray by securely taping up the ends.
SECTION: Making the Gel
10. Once the agarose has cooled (5-10 minutes should be OK), add 4 µL of   to the cooled gel mixture and swirl to mix. 
 


SECTION: Making the Gel
11. Pour the molten agarose into the gel tray and insert the comb (make sure it is straight and level) and leave to set.
SECTION: Making the Gel
3. Weigh out the correct amount of agarose and place it in a 250 mL conical flask. Note: for a 1% gel you will need 1 g of agarose for every 100 mL of buffer.

SECTION: Loading and Running the Gel
12. Add TAE buffer to the tank, then add the gel and ensure it is fully submerged in TAE. 

SECTION: Loading and Running the Gel
13. Load the DNA samples into the gel, and add a DNA ladder to one of the wells
SECTION: Loading and Running the Gel
14. Run the gel at 100V for 60 min

SECTION: Loading and Running the Gel
15. Observe the gel and locations of the DNA fragments
TITLE: JAX-Sen: Mice selection criteria
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
We aim to select 12 mice (C57BL/6J), 6 male and 6 female at 6 and 24 months of age, 3 mice in each age and sex group. This number is for each analysis (scRNA-seq, snRNA-seq, bulkRNA-seq, and visium) for a total of 48 animals.

[STEPS]
TITLE: Protein A- Protein A Sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study Staphylococcal protein-A (SpA) interactions with various mammalian and avian immunoglobulins.</div></div>

[STEPS]
?. This ELISA was used to study Staphylococcal protein-A (SpA) interactions with various mammalian and avian immunoglobulins. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpA in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. Fifty microliters of immunoglobulins (1 mg/mL) was added and incubated for 1.30h at room temperature, and the microplate was then rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled-SpA conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1.30h at RT. The plate was washed 4X with PBS-Tween.
?. Fifty microliters of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) was pipetted into each well. The reaction was stopped with 50 µL of a 3M H2S O4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 450 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The higher the OD value, the higher the affinity of SpA for avian immunoglobulins. The cut-off point was set to 0.30.
TITLE: The effectiveness of high intensity exercise therapy for improving bone density in postmenopausal women: a systematic review and meta-analysis protocol
AUTHORS: Takashi Kitagawa, Kaede Hiraya, Homare Hirokawa, Takumi Denda, Nanako Otake, Yoshiki Saimon, Shuhei Yamamoto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The purpose of the current review is to examine the effects of high intensity exercise on bone mineral density (BMD) changes compared to moderate intensity exercise, and how the safety of exercise differs. We will cover randomized controlled trials assessing the effectiveness of high intensity exercise therapy for improving bone density in postmenopausal women. To determine the difference in effect on BMD changes at the lumbar spine and femoral neck between high intensity exercise therapy and moderate intensity exercise therapy in postmenopausal women (P: postmenopausal women, I: high intensity exercise therapy, C: moderate intensity exercise therapy, O: BMD changes at the lumbar spine and femoral neck). Six databases will be searched in this review. After screening the studies, two reviewers will evaluate the risk of bias independently using the Risk of Bias 2. To evaluate statistical heterogeneity, we will visually inspect the forest plots and calculate the I</span><span style = "vertical-align:super;">2</span><span> statistic. Meta-analysis will be carried out with Review Manager software (RevMan 5.4), using a random-effects model. Summary of findings table will be generated.</span></div></div>

[STEPS]
TITLE: Preparing 10X Genomics CytAssist FFPE Sections
AUTHORS: YuHong Fu, Glenda Halliday

[DESCRIPTION]
Designing and properly mounting FFPE sections on large-window CytAssist slides is important for downstream analysis and data quality. This protocol describes the key steps for this preparation. 

[STEPS]
SECTION: Set-up before cutting FFPE sections
1. Clean the surface of the bench area, microtome (HistoCore MULTICUT R, Leica), Water bath (HI1210, Leica), and tools (including forceps and blades) with 80% ethanol.
SECTION: Set-up before cutting FFPE sections
2. Spray RNase ZapTM (Thermo Fisher Scientific) on the surface of the bench area, instruments, and tools and leave them to completely dry before use.
SECTION: Set-up before cutting FFPE sections
3. Filled the Water bath with RNAse/DNAse free water and set the temperature at 40℃.
SECTION: Cut FFPE sections
4. Cut FFPE sections at 6um thickness.
Note: The time cost indicated for this step is for one FFPE block.
SECTION: Cut FFPE sections
5. Mount sections in the center region (< 11mm x 11mm) of the poly-prep slide according to the 10X genomics analysis requirement. 
Note: The time cost for this step is experiment task-dependent.
SECTION: Cut FFPE sections
6. Incubated mounted sections at 42°C for 3 h and dried overnight at room temperature in a desiccator. 
SECTION: Cut FFPE sections
7. Ready for downstream CytAssist procedure.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Extraction of genomic DNA from Arabidopsis leaf material.</p>

[BEFORE_START]
<p>Make CTAB buffer:</p>
<p>- 2% (w/v) CTAB</p>
<p>- 1.4 M NaCl</p>
<p>- 0.1 M Tris-HLC pH 8</p>
<p> </p>
<p>Grind leaf tissue into fine powder using mortar and pestle or Qiagen tissue lyser (place 1/8" steel ball bearing into tube with tissue sample).</p>

[GUIDELINES]
<p>Gives reasonable quality and yield of gDNA, typically would use for PCRs, sequencing, and cloning but not for next-generation sequencing.</p>

[STEPS]
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