eamag/protocols-with-steps huggingface dataset:
TITLE: 0.1xBWT+SDS buffer
AUTHORS: Anna Schmidt, Sarah Nagel, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of 0.1x BWT+SDS buffer (Bind and wash buffer I) for automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020).

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.

[STEPS]
TITLE: The efficacy of telemedicine using videoconferencing system in outpatient care for cancer patients: a systematic review and meta-analysis protocol
AUTHORS: Yasuaki   Uemoto, Taro Yamanaka, Yuki  Kataoka, Yoshitaka  Wada, Yosuke Aoyama, Rika Kizawa, Yu Yamaguchi, Yuichiro Kikawa, Hirohumi Mukai, Naruto Taira

[DESCRIPTION]
Further research is needed to compare the impact of interactive Telemedicine using videoconferencing systems with usual face-to-face care in the outpatient with cancer. The aim of this study is to review systematically the efficacy of interactive telemedicine using videoconferencing systems in this setting, including feasibility, satisfaction and cost.

[STEPS]
TITLE: SW-LB Media (1L)
AUTHORS: Brian Smith, baltrus@email.arizona.edu micro.tucson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will make one liter of salt water LB.  Adjust volumes as necessary.</div></div>

[STEPS]
?. [Fill container aproximately 750mL of H2O]
Add stir bar and set to mix
?. [Add Ingredients]
[Bacto Tryptone]
[Yeast Extract]
[NaCl]
[MgSO4•7H2O]
[NaHCO3]
?. [Bring volume to 1L by adding H2O]
Allow to mix again with stir bar.
?. [Add Agar (if making plates)]
Add agar to container to be autclavedThen add media.
[agar]
TITLE: Analyzing oxygen consumption of isolated mitochondria using the Seahorse XFe96 analyzer
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for analyzing oxygen consumption of isolated mitochondria using the Seahorse XFe96 analyzer.

[STEPS]
SECTION: Day 1
1. Seed cells in 10 cm plates aiming for a confluency of ~80-90% at the time of treatment.
SECTION: Day 1
2. Add 200 µL of Seahorse XFe calibrant solution to each well of a Seahorse (Agilent) cartridge plate, and incubate   at 37 °C in a CO2-free incubator.

SECTION: Day 2
3. Isolate mitochondria (see previously published protocol) with the following modifications: at harvesting, scrape cells into ice cold modified isolation buffer (70 mM sucrose, 210 mM mannitol, 1 mM EGTA, 0.5% w/v BSA (fatty acid free), 5 mM HEPES pH 7.2), store cell pellets on ice prior to homogenization, store mitochondrial samples on ice, and immediately assay mitochondrial samples after quantification (frozen mitochondrial stocks cannot be used for oxygen consumption analysis).
SECTION: Day 2
4. Quantify mitochondria by bicinchoninic assay
SECTION: Day 2
5. Aliquot out 15 µgof mitochondria per sample, diluting each aliquot to a final volume of 25 µL in mitochondrial assay solution (MAS: 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl26H2O, 1 mM EGTA, 0.1% w/v BSA (fatty acid free), 2 mM HEPES pH 7.2), and leave the samples on ice until needed.

SECTION: Day 2
6. Pre-chill a Seahorse sample plate on ice
SECTION: Day 2
7. Make up the substrate solution (10 mM Glutamate, 10 mM malate in MAS buffer) and place at 37 °C in a CO2 free incubator for at least60 min

SECTION: Day 2
8. To the equilibrated cartridge plate, load 20 µL of 20 millimolar (mM) ADP, 22 µL50 ug/uL oligomycin, 24 µL of 10 micromolar (µM) FCCP, 26 µL of 40 micromolar (µM) of Antimycin A into the corresponding ports of each well.

SECTION: Day 2
9. Incubate the cartridge at 37 °Cin a CO2-free incubator for 45 min, and begin the calibration sequence on the Seahorse XFe96 analyser so that it’s completion corresponds with step 13 (takes ~30 min). 

SECTION: Day 2
11. Centrifuge the plate at 2000 rcf, 20 min, 4 °C, and place the plate on ice until required. 



SECTION: Day 2
10. During step 9, add a 25 µL aliquot of mitochondria to each corresponding well of the pre-chilled Seahorse sample plate.

SECTION: Day 2
12. Add 155 µLof pre-warmed substrate solution to each sample well.

SECTION: Day 2
13.  Eject the calibration plate from the analyser and replace it with the sample plate.
SECTION: Day 2
14. Run the following protocol on the analyser: 
Basal (3 min mix, 3 min measure, 3 min mix, 3 min measure)
ADP (injection, 30 sec mix, 3 min measure)
Oligomycin (injection, 30 sec mix, 30 sec wait, 3 min measure)
FCCP (injection, 20 sec mix, 3 min measure)
Antimycin A (injection, 30 sec mix, 3 min measure)
SECTION: Day 2
15. If analyzing mitochondrial respiration across multiple days, perform step 2 of day 1 the day before the time point analysis, and perform step 3 – 14 on each day of the time course with the appropriate vehicle-treated controls each day.
TITLE: Hot Water Tail Immersion Test 
AUTHORS: Lani Tieu, Brent Boomhower, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The tail immersion test measures pain response (tail flick latency) to thermal stimuli, and can be used to evaluate the effectiveness of and tolerance to analgesics (e.g. oxycodone).</div></div>

[STEPS]
?. [General Procedure]
Pick up rat from its home cage and wrap it in a towel or pee pad to restrain it, leaving tail exposed.
?. [General Procedure]
The dipper will hold the restrained animal over the water bath and count down before dipping 1 cm of the distal tail into the water. As soon as the tip of the tail is submerged, the timer will start the stopwatch to begin timing the rat's tail flick latency.
Animal must be relaxed with its tail being straight and still before immersing it in the bath.
?. [General Procedure]
As soon as tail flick reflex is observed, the timer will stop the stopwatch and record the tail flick latency.
Tail flick reflex is indicated as animal attempting to withdraw its tail from the water (i.e. flicking the tip of its tail up).
Do not allow more than 10 seconds to pass without observing a tail flick. Finish the procedure and record the time as 10 seconds to avoid tail damage.
?. [General Procedure]
Dry the rat's tail and return it back to its cage.
?. [Drug Onboard Procedure (for IV oxycodone studies in rats)]
Inject 6 animals with 0.3 ml oxycodone (0.15 mg/kg) via the IV catheter.
?. [Drug Onboard Procedure (for IV oxycodone studies in rats)]
Begin a stopwatch upon infusion of the first animal:Every 5 minutes, inject another 6 animals as in Step 1 and repeat this step for all animalsAfter 15 minutes, begin the above tail immersion procedure for the first 6 animals in the order of drug infusion
?. [Drug Onboard Procedure (for IV oxycodone studies in rats)]
Perform tail immersion procedure on each successive set of animals every 5 minutes.
Work efficiently! 5 minutes should be just enough time to complete testing for each set of animals.
TITLE: Bottom agar: 1.5% w/v LB(Miller) agar
AUTHORS: HANNAH ZHU

[DESCRIPTION]
How to make the bottom agar for plaque assay experiment

[STEPS]
1. 
 
 Dissolve following components in 1 L MilliQ water
2. Autoclave at 121 °C for 15 min

3. Pour onto petri dishes (approximately 20 mL per plate (10 cm diameter plates)).

4. Store at 4 °C
TITLE: Tissue dissociation protocol for Pan Immune Project Tissue
AUTHORS: Lira Mamanova

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

[STEPS]
?. Chop tissues into small pieces, weigh
?. Add
?. Run on GentleMACS Octo 30 mins/37oC cycle
?. Stop liberase with 2mM EDTA
?. Mash through smart strainer
?. PBS wash/spin cells to pellet at 500g/10 minutes. Resuspend pellet in PBS.
?. Layer onto ficoll and spin at RT 400g/25 minutes
?. Remove MNCs from ficoll later and wash with PBS
?. Filter through 0.2uM filter
?. Count viable cells and resuspend at concentration of 1000/uL in 10X buffer (chilled PBS + 0.05% FBS) for loading to 10X
TITLE: EMCCD Gain and Offset Calibration 
AUTHORS: Joseph S Beckwith

[DESCRIPTION]
EMCCD Gain and Offset Calibration

[STEPS]
SECTION: EMGain 0 Camera gain and offset measurement protocol
1. Place a lens tissue on the sample stage above the objective lens to serve as a diffuser.
SECTION: EMGain 0 Camera gain and offset measurement protocol
2. Acquire brightfield mode images from the microscope with a 100ms exposure time, capturing at least 500 frames consecutively under no light. Ensure EM Gain is off.
SECTION: EMGain 0 Camera gain and offset measurement protocol
3. Repeat step 2 with increasing exposure times (at least 5 times, including step 2).
SECTION: EMGain 0 Camera gain and offset measurement protocol
4. Save the images taken under different exposure times in the 'camera_calibration' folder in .tif format and prefix the file names with 'exposure_'. For instance, the first image captured under no light should be named '0_filename', and the second image taken with slightly increasing exposure should be named '1_filename'.
5. 
SECTION: EMGain on Camera gain and offset measurement protocol
6. Acquire brightfield mode images from the microscope with a 100ms exposure time, capturing at least 500 frames consecutively under no light. Ensure EM Gain is set to the setting of interest (e.g. 250).
SECTION: EMGain on Camera gain and offset measurement protocol
7. Repeat step 2 with increasing exposure times (at least 5 times, including step 2). Ensure no pixels are above/at saturation.
SECTION: EMGain on Camera gain and offset measurement protocol
8. Save the images taken under different exposure times in the 'camera_calibration_EMGain_N' folder in .tif format and prefix the file names with 'exposure_'. For instance, the first image captured under no light should be named '0_filename', and the second image taken with slightly increasing exposure should be named '1_filename'.
SECTION: Gain calculation
9. Use the GDSC-SMLM plugin for imageJ to calculate gain at no gain, ("Mean-Variance Test") to calculate camera gain.

Then, using this gain parameter, use the "Mean-Variance Test (EM-CCD)" function to calculate the overall game and offset.
TITLE: Refine 365 Keto Gummies Reviews -Is It Scam or Legit?
AUTHORS: Refineketogummy  

[DESCRIPTION]
Refine 365 Keto Gummies Reviews -Is It Scam or Legit?


[STEPS]
1. Refine 365 Keto Gummies Reviews -Is It Scam or Legit?
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This Agar medium can be used for the cultivation of Phytophthora infestans. It is particularly suitable to induce sporulation of Phytophthora on plate.</p>
<p>For long term storage on plate, Rye B agar is recommended.</p>
<p> </p>
<p>This protocal has been copied from http://www.plantpath.cornell.edu/Fry/</p>
<p> </p>
<p>Original reference: Caten, C. E. and J. L. Jinks. 1968. Spontaneous variability of single isolates of<em> Phytophthora infestans</em>. I. Cultural variation. Can. J. Bot. 46: 329-348.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Creating Tensor Maps
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will provide a basic guide creating Tensor maps.</div><div class = "text-block">Note: Steps may vary based upon data.</div></div>

[STEPS]
?. [Introduction]
This protocol will present the steps needed to compute multiple tensor maps on the HPC in TORTOISE. The protocol assumes that DIFFPREP and DRBUDDI have already been applied.
?. [Create Mask from T2 Structural]
Open up ITK-snap to create mask.Type the following in a terminal:
?. [Create Mask from T2 Structural]
Once ITK-SNAP opens browse to the structural image.The folder you should be pulling the structural.nii image from is in DTITest_DTI_proc_DRBUDDI_proc
?. [Create Mask from T2 Structural]
Click on the snake tool to start making the mask.
?. [Create Mask from T2 Structural]
Move the red lines to adjust boundaries for the fill function.
?. [Create Mask from T2 Structural]
Select Segment 3D after you finish adjusting the red lines.
?. [Create Mask from T2 Structural]
Select the blue thresholding view and then adjust your thresholding till you are satisfied.
?. [Create Mask from T2 Structural]
For thresholding typically you want to remove bone, muscle tissue, and eyes if possible.
?. [Create Mask from T2 Structural]
Click the Add Bubble at Cursor button while clicking around on all the 3 different views to move the cursor.
?. [Create Mask from T2 Structural]
Select Next in the lower right hand corner.
?. [Create Mask from T2 Structural]
Select the play button to grow the bubbles.You can additionally select the update button at the bottom of the screen to view your growing mask.
?. [Create Mask from T2 Structural]
Once you have added enough "bubbles" select next.
?. [Create Mask from T2 Structural]
When the mask is done select the pause button and click finish.
?. [Create Mask from T2 Structural]
You can select update a second time and your completed mask will appear in the lower left hand corner.
?. [Create Mask from T2 Structural]
Save your mask as a NIFTI file, and exit out of ITK-SNAP.
?. [Estimate Tensor]
Type the following to get information about the command.
?. [Estimate Tensor]
Type the following:
?. [Estimate Tensor]
This creates two files a _DT.nii and a _AM.nii. You will use the _DT.nii file.
?. [Compute Tensor Map]
To create every tensor map type the following command:
?. [Compute Tensor Map]
The above command runs a script to create all tensor maps available, but if you want to only create a certain tensor map you can use can just type:This example will only output a DEC map, but if you want to output another map just change the 'DEC' to be the name of the desired map.
?. [Compute Tensor Map]
Open your DEC map in ITK-SNAP.
?. [Compute Tensor Map]
Click on the Layer Inspector button:
?. [Compute Tensor Map]
Choose your display mode to be RGB Display.
?. [Compute Tensor Map]
Below is the finished RGB display of your DEC map.
TITLE: Acetate Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Acetate Buffer]
Mix acetic acid and sodium acetate solutions in the proportions indicated:  ABCDEFGH1mL of Acetic acid46.341.030.520.014.810.54.82mL of Sodium acetate3.79.019.530.035.239.545.23pH3.64.04.44.85.05.25.6
pH range:   to  (a) 0.1 M Acetic acid (5.8 mL made to 1000 mL)(b) 0.1 M Sodium acetate; 8.2 g L-1 (anhydrous; M.W. 82.0 g mol-1) or 13.6 g L-1 (trihydrate; M.W. 136.0 g mol-1)
ABCDEFGH1mL of Acetic acid46.341.030.520.014.810.54.82mL of Sodium acetate3.79.019.530.035.239.545.23pH3.64.04.44.85.05.25.6
?. [Acetate Buffer]
Adjust the final volume to  with deionized water.
100 ml
?. [Acetate Buffer]
Adjust the final pH using a sensitive pH meter.
TITLE: RPA DNA Amplification using Agdia AmplifyRP® Acceler8® Discovery Kit
AUTHORS: Théo Nass, Luc Gabel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure to perform an RPA DNA Amplification using the Agdia AmplifyRP® Acceler8® Discovery Kit</div></div>

[STEPS]
?. Prepare the RPA pellet rehydration solution following the table in the Materials section.If possible do a Master Mix that will then be divided.
?. For each sample, transfer the entire volume (10 µl) to a reaction pellet. Pipette up and down until the full pellet has been resuspended.
?. Cap the reaction tubes. Vortex and spin briefly two times.
?. Transfer the reaction tubes to a heat block. Incubate at 39°C for 20 min.
?. After the incubation, purify the sample if you want to run the RPA product on an agarose gel.PCR purification kits work well, but since RPA primers can be very long (60+ bp), a high cutoff is recommended.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol series will guide students through the experience of analyzing metagenomic data. 

[STEPS]
SECTION: DNA quality assessment and assurance
?. 
SECTION: Metagenomic assembly
?. 
SECTION: Assessing the quality of the assemblies
?. 
SECTION: Binning assembled metagenomes with MaxBin
?. 
SECTION: Assessing the quality of your bins via CheckM
?. 
SECTION: Assuring DNA sequencing quality using Trimmomatic
?. 
SECTION: Use VizBin to further curate your bins
?. 
SECTION: Perform taxonomic identification using Phylosift
?. 
SECTION: Prokka - software for annotations
?. 
SECTION: Compare genomes to various databases
?. 
SECTION: Compare genomes to various databases
?. 
SECTION: Compare genomes to various databases
?. 
SECTION: Running Phylosift on metagenomic reads using tmux
?. 
SECTION: Running PROKKA and COG on your metagenomic contigs
?.
TITLE: Immunofluorescence Assay (IFA)
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for performing an immunofluorescence assay with fixed HeLa cells.

[STEPS]
SECTION: Day 1
1. Wash HistoGrip (ThermoFisher) coated coverslips 1x in sterile PBS.
SECTION: Day 1
2. Seed cells in 6 well plate wells containing HistoGrip (ThermoFisher) coated coverslips in standard growth media, aiming for a confluency of ~70-80% at the time of treatment the next day.
SECTION: Day 2
3. Feed cells for 60 min prior to treatment with the desired growth compounds in standard growth media.

SECTION: Day 2
4. At the conclusion of the treatment, aspirate all media from the well and replace with 500 µL/well of fixative (4 % (v/v) paraformaldehyde in 0.1 Molarity (M) phosphate buffer). Incubate for 10 min gently rocking at Room temperature.

SECTION: Day 2
5. Remove the fixative, and replace with 500 µL/well of permeabilization buffer (0.1 % (v/v) Triton X-100 in PBS). Incubate for 10 min gently rocking at Room temperature.

SECTION: Day 2
6. Aspirate the permeabilization buffer, and replace with 500 µL/well of blocking buffer (3 % (v/v)goat serum in 0.1 % (v/v) Triton X-100 in PBS). Incubate for 15 min gently rocking at Room temperature. 

SECTION: Day 2
7. During step 4, make up the primary antibody solutions containing the desired antibodies in blocking buffer. 
SECTION: Day 2
8. Aspirate the blocking buffer, and wash each well 3x with PBS. 
SECTION: Day 2
9. Wash each well once with blocking buffer, and when aspirating the blocking buffer, gently nudge the coverslip into the center of the well. 
SECTION: Day 2
10. Using a p200 pipette, carefully add 25 µL of the primary antibody solution directly onto the cover slip of each well. Ensure the bench this is performed on is flat. Replace the lid on each plate and cover all plates from light.

SECTION: Day 2
11. Incubate samples in the primary antibody solution for 120 min at Room temperature (static, no rocking). During this incubation, make up the secondary antibody solutions containing the desired antibodies in blocking buffer. Cover this solution to protect it from light until it is needed.  

SECTION: Day 2
12. Wash each sample 3x in PBS. 
SECTION: Day 2
13. Wash each well once with blocking buffer, and when aspirating the blocking buffer, gently nudge the coverslip into the center of the well. 
SECTION: Day 2
14. Using a p200 pipette, carefully add 25 µL of the secondary antibody solution directly onto the cover slip of each well. Ensure the bench this is performed on is flat. Replace the lid of each plate and cover all plates from light.

SECTION: Day 2
15. Incubate samples in the secondary antibody solution for 120 min at Room temperature (static, no rocking). 

SECTION: Day 2
16. Wash the samples 3x in PBS
SECTION: Day 2
17. Add PBS to each well to keep the coverslips hydrated prior to mounting.
SECTION: Day 2
18. Mount coverslips onto glass slides using a small amount of TRIS-buffered DABCO-glycerol mounting medium.
SECTION: Day 2
19. Seal the edges of each coverslip with black nail polish, and once dry, store slides protected from light at 4 °C until imaging.