eamag/protocols-with-steps huggingface dataset:
TITLE: Diamond XChem Seeding Protocol
AUTHORS: Peter Marples, daren.fearon, Charlie Tomlinson

[DESCRIPTION]
The use of seeding in protein crystallisation is a highly effective method for increasing the quantity of usable crystals from a single plate and can be crucial for obtaining crystals in a highly reproducible  manner. Even crystallisation experiments which did not previously require seeding can benefit from  the use of seeds when transferring protocols between labs.

[BEFORE_START]
Prepare crystalline material needed to produce crystals.

[GUIDELINES]
N/A

[STEPS]
SECTION: Equiptment needed
1. Formulatrix Protein Crystallization Imager
SPT mosquito 
P10 pipette
SwissCI 3 lens plate [UVXPO-3LENS3W96T-PS3W96T-UVP]
Seed beads [Hampton HR4-782]
Crystalline material
Vortex
0.5 mL Eppendorf tubes [Eppendorf Catalog No. 0030121023]
SECTION: Seeding experiment
2. Produce crystals to use for seeding. Micro crystalline protein material can be used to produce a seed stock as well.
SECTION: Seeding experiment
3. Set up the serial dilution for step 10 using the reservoir solution used to obtain the starting crystalline material on ice. Make up an appropriate volume; most seeding uses 20-50 nL of seed stock, meaning a SwissCI 3 lens 96 well plate could require ~14.5 µL of seed stock per plate.

SECTION: Seeding experiment
4. Add seed beads to the undiluted tube in the serial dilution (Check manufacture instructions).
For Hampton glass beads, use 2 beads in a 0.5 mL Eppendorf tube
SECTION: Seeding experiment
5. Under a microscope, open the well containing the crystal material being used and crush the crystals. From this step onwards proceed rapidly as the seed stocks are metastable and need to be frozen as soon as possible.
SECTION: Seeding experiment
6. Pipette2 µL from the reservoir well into the drop well

SECTION: Seeding experiment
7. Mix the solution by aspiration a minimun of three times
SECTION: Seeding experiment
8. Transfer to the first tube set up for the serial dilution that will remain undiluted and is marked "undiluted seed stock"
SECTION: Seeding experiment
9. Repeat steps 5-7 until there is no more crystal in the drop well. To ensure all available crystal material is recovered, a minimum of around 30 µL of reservoir solution should be used in this process

SECTION: Seeding experiment
10. Vortex the undiluted tube containing the seed beads – 30 seconds on vortex then 30 seconds on ice, repeating three times. Take 2 µL of the seed stock and check under microscope whether crystals have been crushed.

SECTION: Seeding experiment
11. Carry out the set up serial dilution using the undiluted seed stock and freeze stocks at -80 °C

SECTION: Interpreting results
12. Based on what your project needs, decide which condition made from the serial dilution makes the best results with good reproducability. If needed, further optimise around the dilution e.g. if 1/100 works best, try using 1/20, 1/50 and 1/200; repeat until crystals are acceptable for your project and its timeframe.
TITLE: 11 Concentration and fluid exchanging
AUTHORS: TJUSLS China

[STEPS]
?. [Concentrate]
1. The default concentrator is already inactive.2. Pre-cool the Thermo centrifuge to 4 °C in advance.3. Pour the protein solution to be concentrated into a concentrating tube, level it with the trim tube, place it in a Thermo centrifuge at 3400 rpm, stop at half an hour, pour off the effluent, and add the protein solution to the upper layer.4. If the protein is used for long crystals, it can be concentrated to less than 500 μl. According to the amount of protein and protein performance, it is determined that the protein is colloid rather than turbid liquid. If the protein is used for 24 ml molecular sieves, concentrate to within 1 ml.5. Pipet the concentrated protein into a Ep tube with a 200 μl pipette (with a yellow tip).
4 °C
200 µl
?. [Disposal of the reuse of the concentrating tube]
1. Rinse the upper chamber of the used concentrator tube with distilled water several times, and add some hypersaline solution overnight at room temperature.2. Pour off the sodium hydroxide from the upper chamber, rinse several times with distilled water, and centrifuge 3000 rpm for 5 minutes with distilled water.3. Step 2 is repeated 3 times.4. Add 20% ethanol or ddH2O to the upper chamber and store at 4 °C.
4 °C
?. [change the liquid]
The steps are the same as the concentration, and the buffer is added without any what.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is kit-free and can be used to isolate high quality genomic DNA of predominantly <em>Symbiodinium</em> from symbiotic Aiptasia which can be used as e.g. PCR template for genotyping.</p>
<p> </p>
<p> It is based on the method described in Coffroth et al., 1992.</p>

[BEFORE_START]
<p>Recipes:</p>
<p><strong>2×CTAB Buffer</strong></p>
<p><em>(Coffroth et al. (1992) Marine Biology 114: 317-325)</em></p>
<p> </p>
<p><strong>End concentrations:</strong>                                             <strong>For 100 ml buffer mix:</strong></p>
<p>1.4 M              NaCl                                                            28 ml of a 5 M stock solution</p>
<p>20 mM            EDTA (pH 8)                                                 4 ml of a 0.5 M stock solution</p>
<p>100 mM          Tris/HCl (pH 8)                                          10 ml of a 1 M stock solution</p>
<p>2% (w/v)        CTAB powder                                               2 g</p>
<p><u>Add after filter sterilization under a hood:</u></p>
<p>0.2% (v/v)      β-mercaptoethanol (<strong><u>TOXIC!!!</u></strong>)              200 µl</p>
<p> </p>
<ul>
<li>Add ddH<sub>2</sub>O to just under 100 ml.</li>
<li>Warm to 65°C under stirring to bring the CTAB into solution.</li>
<li>Once dissolved, bring final volume to 100 ml using a graduated cylinder.</li>
<li>Filter sterilize (0.2 µm) into sterile 50 ml Falcon tubes and store at −20°C.</li>
</ul>
<p>Heat to 65°C before usage since freezing leads precipitation of the CTAB.</p>

[GUIDELINES]
<p>Make sure to use Phenol:Chloroform:Isoamyl alcohol (25:24:1) which is buffered with TE to a neutral to slightly basic pH (everthing starting from 6.7 should work) since DNA stability decreases at acidic pH (the opposite is true for RNA).</p>
<p> </p>
<p>Be sure not to exceed the maximal ×g for which your microcentrifuge tubes are certified otherwise they might get damaged during centrifugation and release all the hazardous substances (and the DNA of course) into the rotor. You don't want that!</p>
<p> </p>
<p>If making your own stock solutions of RNase A from powder be aware that some products contain DNase impurities. If this should be the case you need to boil the stock solutions once for 15-20 min at 100°C and let cool down at room temperature to inactivate the DNases. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Use of a FlowCam for descriptive measurements of Aureococcus anophagefferens live culture cells
AUTHORS: Emily E. Chase, Laura Smith, Alex Truchon, Steven W Wilhelm

[DESCRIPTION]
A protocol for acquiring an array of descriptive statistics (e.g., cell count, cell diameter, cell volume) for a monoculture of Aureococcus anophagefferens. This procedure permits an in-depth profile of your cultures via a substantial number of viewable photos and measurements. We recommend this familiarity with your study cells, especially when applying stress treatments such as virus infection, light cycle changes, etc. 

[BEFORE_START]
Familiarise yourself with the functions of the FlowCam equipment and Visual Spreadsheet5 software. 

[STEPS]
SECTION: SAMPLE AND CONTEXT FILE PREPARATION
2. Aureococcus anophagefferens cells are retrieved from a culture for measuring. At least 500 μL are required for each sample. 

 



SECTION: SAMPLE DESCRIPTIVE STATISTICS ACQUISITION
4. Following set-up of the FlowCam by manufacturer's instructions (using the 20✕ objective, 20✕ flow cells, and 0.5 or 1.0 mL syringe; green set) and the full clearance of Milli-Q water through the tubing, enter the flow cell view mode (i.e., "Camera View"), apply at least 500 μL of your sample, and start the flow at 1–2 mL/minute. Once cells appear within the on screen camera aim at the flow cell, halt the manual priming (close the window). 


 

SECTION: SAMPLE AND CONTEXT FILE PREPARATION
3. Open the Visual Spreadsheet5 software, and load the appropriate context file. Measuring A. anophagefferens can be accomplished with the following settings in the context file: 
Distance to nearest neighbour: 1.0
Close hole iteration: 2.0
Collage image border padding: 2
Dark pixels: 20.0
Light pixels: 20.0
Acceptable region left: 110; right: 1115; top: 1; bottom: 1919


 

SECTION: SAMPLE DESCRIPTIVE STATISTICS ACQUISITION
5. Cell image capture (and subsequent descriptive statistics) is processed via the AutoImage mode. Simply click the auto image mode, set the acquisition method constraints, provide a sample name/ID, and initiate the sampling.

 

SECTION: SAMPLE DESCRIPTIVE STATISTICS ACQUISITION
6. Record or export relevant data. 

An example of resulting data for A. anophagefferens are provided as Figure 1 and Figure 2 below. 

  
  

 

SECTION: SAMPLE DESCRIPTIVE STATISTICS ACQUISITION
7. Clear the entire FlowCam tubing system of the previous sample, administer a Milli-Q water rinse, and apply a new sample as previously described. Repeat for all samples and then apply FlowCam cleaning as established in your lab. 

 

SECTION: FLOWCAM 8000 OPERATION
1. An explanation of the operating practices of the Flow CAM 8000 are presented under another protocol and through the relevant software and equipment manuals. Equipment setup can be achieved through the linked protocol below.
TITLE: Artificial seawater
AUTHORS: Simon Blanchoud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Three alternative solutions for artificial seawater (ASW) have been tested successfully on our </span><span style = "font-style:italic;">Botrylloides</span><span> colonies. For routine work, we use commercial sea salts (CSS), for most analyses we use the Cold Spring Harbor Protocols (CSPH) and for very clean work the K-depleted Phosphate-buffered saline (K-PBS). While, CSPH and K-PBS can be prepared at 10X, CSS should be prepared at 2X directly.</span></div><div class = "text-block">Mix to dissolve and adjust pH as well as salinity to your local conditions.</div></div>

[STEPS]
?. [2X CSS]
sea salt in  H2O.
70 g
1 L
?. [2X CSS]
Mix to dissolve.
?. [2X CSS]
Filter at 10 µm
?. [10x CSHP]
To prepare 10X CSHP take NaCl262.9g4.5MKCl7.4g100mMCaCl29.9g90mMMgCl2 6H2O60.9g300mMMgSO4 7H2O39.4g160mMH2O1000ml
NaCl262.9g4.5MKCl7.4g100mMCaCl29.9g90mMMgCl2 6H2O60.9g300mMMgSO4 7H2O39.4g160mMH2O1000ml
?. [10X K-PBS]
To prepare 10x K-PBS take NaH2PO4 · H2O2.8 g0.02 MNa2HPO411.4 g0.08 MNaCl87.5 g1.5 MH2O1000ml
NaH2PO4 · H2O2.8 g0.02 MNa2HPO411.4 g0.08 MNaCl87.5 g1.5 MH2O1000ml
TITLE: KAPP-Sen TMC: Placenta tissue blocks paraffin embedding
AUTHORS: Ramalakshmi Ramasamy, Anne Marchini, Tim Adams, Elaine Bechtel, Lesley Bechtold

[DESCRIPTION]
Placenta formalin-fixed tissue blocks paraffin embedding at JAX Bar Harbor Histology Services.

[STEPS]
1. Procedure addition between sample collection at Mayo Clinic and JAX Bar Harbor Histology Services processing:
1.1. Samples were received from Mayo Clinic in 10% NBF on ice.
1.2. Formalin was replaced with 70% Ethanol and left overnight at room temperature.
1.3. Samples were shipped at room temperature to JAX Bar Harbor Histology Services for tissue processing and paraffin embedding (FFPE).
2. At JAX Bar Harbor Histology Services samples were embedded in paraffin (FFPE) according to: https://dx.doi.org/10.17504/protocols.io.rm7vzj9z2lx1/v1
TITLE: DNA - Ball Python DNA Amplification with TFEC primers
AUTHORS: Jose Avila Cervantes

[DESCRIPTION]
PCR amplification with Tfec set Exon 5 to genotype Piedball morph in Ball python (Python regius)

LEFT PRIMER         AACTCAGAGCACTCCATGACC
RIGHT PRIMER       CAGGTGTGCCCCTTTCATAA

[STEPS]
1. REAGENTS
10X PCR Buffer
MgSO420 millimolar (mM) 
Taq Polymerase 5U/ul
Primer Tfec exon5 Left 10 micromolar (µM) 
Primer Tfec exon5 Right 10 micromolar (µM) 
DNTP's 10 micromolar (µM) 
Ultra Pure Water
100bp ladder (100-2,000 bp)
SYBR Safe DNA stain
Loading dye
Agarose

2. EQUIPMENT
DNA LoBind tubes 1.5mL
PCR 8-Strip tubes 0.2mL 
Micropipettes
Thermal Cycler

3. MASTER MIX

Below is the recommended volume for one sample. Adjust for the number of samples to process and add 10% to account for pipetting error. Thaw all reagents at room temperature before using them except the Taq DNA polymerase, which has to be kept on ice at all times. Premix all reagents and add the Taq DNA polymerase at the end, vortex thoroughly for 30 s seconds.  Aliquot the master mix in to each tube 28 µL  and then add 2 µL of DNA sample. The final volume for each reaction is 30 µL .
 
          Volume    10x PCR Buffer  3ul    MgSO4  4.5ul    TAQ  0.3ul    Primer F  1.5ul    Primer R  1.5ul    DNTP's  0.6ul    Template   2ul    Water  16.6  
 













4. PCR CONDITIONS

 



5. AMPLICON QUALITY CHECK

1. Prepare a 1% agarose gel with 1X SYBR Safe DNA stain.

2.  Add  4 µL  of 100bp ladder (100-2,000 bp) in the first well.
 
3.  Premix 4 µL of sample and 1 µL of 10X loading dye or 3 µL of sample and 1 µL of 6X loading dye. Load this mixture in each well.

3. Run the samples for 35 min at a 100V.

4. Visualize in a trans illuminator and take a photo of the gel.

5. The product should be a clear, single band around 200 bp.
TITLE: 2020_cocci_mapping_export_western_USA
AUTHORS: Robert Dobos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This spreadsheet contains map unit agggregated output of the cocci soil habitat model.  Table headers are State, Area Symbol, Area Name, Map Unit Name,  Map Unit Acres, Max HSI, and Mukey.  Mukey is the link to the SSURGO spatial data, which can be obtained from the Geospatial Data Gateway:</div><div class = "text-block"><a href="https://gdg.sc.egov.usda.gov" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://gdg.sc.egov.usda.gov/</span></a><span style = ":;">.</span></div><div class = "text-block">This spreadsheet can be joined to the spatial data using Arc-GIS.  The Mukey field must explicitly be formatted as text.</div><div class = "text-block">The data was extracted from the NASIS transactional database 20 June 2020, and reflect the data present at that time.  The states reported are Arizona, California, Colorado, Idaho, Kansas, Montana, Nebraska, Nevada, New Mexico, North Dakota, Oklahoma, Oregon, South Dakota, Texas, Utah, Washington and Wyoming.  These are the states thought to have at least some area hot, dry, and salty enough for cocci to exist.  This data may be available for some of these states on Web Soil Survey:</div><div class = "text-block"><a href="https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm</span></a><span style = ":;">.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This buffer is used to stabilize samples for archiving and subsequentgenomic DNA extraction. <br /><br />SDS is dissolved to a concentration of 1% (w/v) in DNAB (DNA buffer: 0.4 M NaCl + 0.05 M EDTA in MilliQ water). The buffer may need to be warmed for SDS to completely dissolve. Tissue samples are added to small aliquots of 1% SDS in DNAB and heated to 65°C for 60-90 minutes. They are then stable at room temperature and ready for extraction of genomic DNA.

[GUIDELINES]
SDS is dissolved to a concentration of 1% (w/v) in DNAB (DNA buffer: 0.4 M NaCl + 0.05 M EDTA in MilliQ water). The buffer may need to be warmed for SDS to completely dissolve. Tissue samples are added to small aliquots of 1% SDS in DNAB and heated to 65°C for 60-90 minutes. They are then stablle at room temperature and ready for extraction of genomic DNA.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Morphology of gracilis muscle and the topographic anatomy of its neurovascular pedicles
AUTHORS: chettiar ganesh kumar, Bv Murlimanju

[DESCRIPTION]


Gracilis
is often utilized as a graft in the procedures of reconstruction and functioning
muscle transfer.1 It is excellent for the closure of recto-vaginal
and rectourethral fistulas. The distal pedicle is used during the total
knee arthroplasty as a soft tissue graft.2,3 It also has wider
application in the management of wounds of the groin, contractures, scars,
reanimation of the face,4,5 reconstruction of the mammary gland,6and anterior cruciate ligament reconstruction.7 Additional
harvesting of the gracilis has proven to offer better post-operative functional
activity like in hamstring injury.8 The
main vascular pedicle of gracilis is from the medial and lateral circumflex
femoral arteries, which branch from the deep femoral artery, which enters the
gracilis at the junction of its upper and middle third. Distal part of the gracilis
is supplied by the femoral artery9. 




[STEPS]
SECTION: Morphology of gracilis muscle and the topographic anatomy of its neurovascular pedicles
1. Morphology of gracilis muscle and the
topographic anatomy of its neurovascular pedicles
TITLE: Thawing of cryopreserved hPSC
AUTHORS: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes thawing of hPSC.

[GUIDELINES]
Thawing procedure is described for one cryovial. The protocol description is general and applies to multiple hPSC lines frozen as clumps or single cells. 

It is recommended to thaw hPSC in same culture conditions that were used before freezing.

[STEPS]
SECTION: hPSC thawing
4. Remove cryovial containing frozen hPSC from liquid N2.
SECTION: hPSC thawing
5. Sanitize the tube with 70 % volume ethanol.
SECTION: hPSC thawing
7. Using 2 mL pipette transfer slowly hPSC suspension from the cryovial to a 15 mL conical tube.
SECTION: hPSC thawing
8. Dropwise add 10 mL of culture media supplemented with survival factors to hPSC suspension while gently swirling the tube, refer to Table 1 for recommended volumes. 
SECTION: hPSC thawing
9. Centrifuge hPSC suspension at 300 x g, 5 min
SECTION: hPSC thawing
11. Using a 5 mL pipette gently add 2 mL of hPSC culture medium supplemented with survival factors to the pellet.
SECTION: hPSC thawing
14. Seed hPSC at desired density according to well size/surface.
SECTION: hPSC thawing
15. Rock the plate side to side, back and forth to spread the cells across the well.
SECTION: hPSC thawing
16. Incubate the vessels at 37 °C  and at 5 % volume CO2.
SECTION: hPSC thawing
17. After 1440 min, perform full media change with respective hPSC maintenance media without survival factors.  For further hPSC culture refer to protocol Maintenance of hPSC.
 

SECTION: hPSC thawing
6. Thaw hPSC suspension at 37 °C using water or bead bath for about 2 min -4 min leaving small amount of un-thawed media.
 

SECTION: hPSC thawing
10. Aspirate and remove the supernatant without disrupting the cell pellet.
SECTION: hPSC thawing
12. Gently resuspend the hPSC pellet by pipetting up and down. 
 

SECTION: hPSC thawing
13. Aspirate coating solution from the destination vessel.
SECTION: Preparation of destination vessel
1. Prepare coated tissue culture vessels according to hPSC line requierements and desired format. For coating procedure refer to protocol Coating of tissue Culture Vessels for hPSC
 

SECTION: Preparation of destination vessel
2. Equilibrate prepared destination vessel at 37 °C until usage.
SECTION: hPSC thawing
3. Prepare required volume of the reagents and media supplemented with survival factors according to Table 1.  Additionally prepare 10 mL of media for Step 8. Equilibrate the media to Room temperature or 37 °C.
TITLE: Efficient realization of quantum primitives for Shor&#39;s algorithm using PennyLane library
AUTHORS: Anatoly   Antipov, Evgeniy Kiktenko, Aleksey Fedorov

[DESCRIPTION]
The following protocol describes how to use a software package
containing implementations of various quantum gates and well-known quantum
algorithms using PennyLane library. Templates within the package include all required
elements from the quantum part of Shor’s algorithm, specifically, efficient modular
exponentiation and quantum Fourier transform that can be realized for an arbitrary
number of qubits specified by a user.

[STEPS]
1. First of all, you should install the PennyLane library using the regular python environment and set up the Jupyter Notebook's environment
2. Content of the Github repository should be uploaded on your PC via the following link:

https://github.com/Anatoly-Antipov/QuantumOperations

Files with the extension ".py" represent original python code that realizes the software package with new useful functions built upon the PennyLane library. Functions from this software package are ready-to-use commands similar to PennyLane's functions and commands.

Files with the extension ".ipynb" are Jupyter Notebooks that contain examples of the use of commands from the software package.

See the README.md file in the repository to view an up-to-date description of included files
3. Use the file "Example.ipynb" or the file "ExampleOrderFinding.ipynb" to get the simplest example of the use of a new function. The file "Test.ipynb" contains the full list of newly introduced functions alongside the demonstration of its use in a Jupyter Notebook's environment.