eamag/protocols-with-steps huggingface dataset:
TITLE: PCR Amplification of Desired Gene
AUTHORS: iGEM Dusseldorf

[STEPS]
?. Pipette PCR components in the following order. Always set up at least two replicates.
?. ABCD1# Reactions[µl]Thermocycler ConditionsRounds2Water32.51.       98°C for 0:30 min3Green HF   Buffer10.02.       98°C for 0:10 min5x4dNTPs (10 mM)1.03.       X°C for 0:20 min5F Primer (10   µM)2.54.        72°C for X min6R Primer (10   µM)2.55.       98°C for 0:10 min30x7Template DNA   (10 ng/µL)1.06.       72°C for X min8Phusion DNA   Polymerase0.57.        72°C for 5:00 min9Total50.08.        4°C until use
ABCD1# Reactions[µl]Thermocycler ConditionsRounds2Water32.51.       98°C for 0:30 min3Green HF   Buffer10.02.       98°C for 0:10 min5x4dNTPs (10 mM)1.03.       X°C for 0:20 min5F Primer (10   µM)2.54.        72°C for X min6R Primer (10   µM)2.55.       98°C for 0:10 min30x7Template DNA   (10 ng/µL)1.06.       72°C for X min8Phusion DNA   Polymerase0.57.        72°C for 5:00 min9Total50.08.        4°C until use
?. For step 3, use the annealing temperature of the gene-specific sequences of your primers.For step 4 and 6, adjust the extension time based on the amplicon size (30 s / kb).
?. Prepare a 1 % agarose gel by dissolving agarose in 1x TAE buffer by microwaving (~ 3 min, swirling every min).Add 2.5 µL of 10000x GelRed nucleic acid stain per 50 mL gel and mix well by swirling.When the conical flask is almost cool enough to touch (3-5 min after microwaving), pour slowly in gel tray and then insert the combs.  Use wide-wells for large PCR reaction volumes.Gel will be solidified after 45-60 min. Remove combs and add enough 1x TAE buffer to cover the gel.
?. Load 2-3 µl DNA ladder and whole PCR samplesinto the gel pockets.Connect lid (DNA will migrate to the + electrode). Turn on power supply and run at 80-110 V depending on the thickness and size of the gel.Examine gel after 30-60 min of electrophoresis and run until the marker reached the desired height.Extract bands of the expected height from the gel with and clean up the fragments with an extraction kit.
?. Thermocycler Step 3: 5 cycles with annealing temperature that is equal to the forward or reverse primer with lower Tm. Has to be adjusted for every gene.
?. Termocycler Step 4: Adjust the elongation time with 30 s for each kb.
?. Termocycler Step 6: 30 cylces with 72 °C for elongation and adjusted extention time. Change time with 30 s per kb.
?. If not enough DNA was produced run another PCR after DNA extraction with the purified amplicon as a template and skip the 5 initial rounds.
?. Other optimisation: Increase time to 40 sec/kb
TITLE: Flag co-immunoprecipitation
AUTHORS: michela.deleidi, Pascale Baden

[DESCRIPTION]
Protocol used to pull down V5-FLAG-Gcase interacting proteins in HEK cells

[STEPS]
SECTION: Flag co-immunoprecipitation
1. Detach HEK cells using Accutase for 5 minutes at 37°C and collect them.
SECTION: Flag co-immunoprecipitation
2. Spin cells in a centrifuge at 250g for 5 minutes at room temperature.
SECTION: Flag co-immunoprecipitation
3.  Remove the supernatant and wash cells in PBS.

SECTION: Flag co-immunoprecipitation
4. Repeat steps 2 and 3 for a total of 2 washes.
SECTION: Flag co-immunoprecipitation
5. Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)
SECTION: Flag co-immunoprecipitation
6. Filter lysates through a 0.22uM PES syringe filter (Millipore).
SECTION: Flag co-immunoprecipitation
7. Wash 50ul of M2 antiflag agarose resin (Sigma–Aldrich, #A2220) according to the manufacturer's instructions for 10 mg of total protein.
SECTION: Flag co-immunoprecipitation
8. Incubate 10 mg of lysates with prewashed M2 anti-flag agarose resin for 2h at 4°C on a rotating wheel.
SECTION: Flag co-immunoprecipitation
9. Elute Flag with Flag Peptide (Sigma-Aldrich, #F3290) by moving on a wheel for 20 min at 4°C followed by a spin for 1 min at 1000 g.
SECTION: Flag co-immunoprecipitation
10. Collect the flow-through and proceed with TMT labeling or Western Blot analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adapted from: Ortmann and Suttle (2009) <em>Determination of Virus Abundance by Epifluorescence microscopy</em>. Ch. 10 Methods of Molecular Biology.</p>
<div>
<div>
<div data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">Contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Samantha Coy (srose16@vols.utk.edu) for additional information regarding this protocol.</div>
</div>
</div>
<div> </div>

[STEPS]
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TITLE: GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
AUTHORS: Tina Lusk Pfefer, Julie Haendiges, Maria Balkey, Candace Hope Bias, Ruth Timme

[DESCRIPTION]
Here we have created a collection of all the protocols used for WGS using the MiSeq, in order, from sample extraction to NCBI submission. 

This collection has three sections:

Section 1: WGS Wet lab workflow for Illumina MiSeq
Section 2: Dry lab workflow for sequence QC and NCBI submission - Direct Submission
Section 3: Dry lab workflow for sequence QC and NCBI submission - PulseNet labs

Version history: 
V3: 
Links to protocols updated with latest version,
Added new protocol: DNA Extraction from Inoculated Broth for WGS using the Maxwell RSC Cultured Cells DNA Kit,
Added new protocol: Querying for Bacterial Pathogen Genomic Data at NCBI.

[STEPS]
SECTION: WGS Wet Lab Workflow for Illumina MiSeq
1. DNA Extraction - Manual
 

SECTION: WGS Wet Lab Workflow for Illumina MiSeq
3. DNA Quantification
 

SECTION: WGS Wet Lab Workflow for Illumina MiSeq
4. Library Preparation

 

SECTION: WGS Wet Lab Workflow for Illumina MiSeq
5. Sequencing
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
6. The following protocols are also included in a Springer Methods book chapter collection: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback (protocols.io)

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
7. Check sequence quality:
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
8. Populate BioSample AND SRA metadata templates:
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
9. Submit sequence and metadata to NCBI:
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
11. Update, retract, or replace your submissions at NCBI:
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
12. Check sequence quality:

Follow the CDC's guidance for assessing QC in BioNumerics
SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
13. Populate NCBI template: 

***NOTE: For NCBI submission for fields not included in the BioNumerics Template, please remember to include the name of the laboratory sequencing the isolates and the surveillance effort name in the sequenced_by and project_name fields as described in Step 5 of this protocol.

 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
14. Submit sequence and metadata to NCBI:

Follow the CDC's guidance for NCBI submissions through BioNumerics.
SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
16. Update, retract, or replace these records in NCBI databases, if necessary:

  
SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
10. Querying data at NCBI
 

SECTION: Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
15. Query data at NCBI

 
SECTION: WGS Wet Lab Workflow for Illumina MiSeq
2. DNA Extraction - Automated
TITLE: Protocol for the purification of total DNA from soil samples
AUTHORS: Ruslan Kalendar

[DESCRIPTION]
The protocol can also be used for other 'complex' samples (e.g. faeces) and tissues containing high amounts of polymeric molecules, pigments that cannot be separated in any other way. The scale in this example is taken to obtain DNA from 1 - 5 grams samples but can be scaled to microvolume and sample mass. 
The goal of this protocol is to isolate as much total DNA from the sample as possible, without purification. The sample can be soil or any other source for which all existing approaches for isolation and purification are unsuitable. Once the DNA sample is in a soluble and concentrated form, it can be further purified by other approaches.

[STEPS]
SECTION: Lysis
6. The tubes are centrifuged at +4С for 10 minutes at 10,000 rpm and any supernatant is discarded. A brown DNA precipitate containing soil components was formed at the bottom of the tube. 
SECTION: Grinding
1. The sample is ground at -80°C by freezing in liquid nitrogen or at -80°C and ground in a mortar or stirrer chilled to -80 °C. 
SECTION: Lysis
4. Soil sample tubes are centrifuged to clarify and precipitate soil, in a large tube centrifuge, at +4°С, for 10 minutes at 10,000 rpm.   
 ,,







SECTION: Lysis
5. The clarified supernatant is separated into two equal portions of 20 ml each and transferred into new 50 ml Falcon tubes into which an equal volume of 100% isopropanol (20 ml) cooled to -20°С has already been added and mixed vigorously on a stirrer. 
SECTION: Lysis
7. 10 ml of 70% ethanol is added to the Falcon tubes with the pellet, and the precipitate is well mixed on a stirrer. The tubes are centrifuged at +4С for 3 minutes at 10,000 rpm. 
SECTION: Lysis
8. The supernatant alcohol solution is completely removed, not dried, and 1 ml of 1x TE solution is added immediately to the precipitate, stirred well, and incubated at 65С until the DNA precipitate is completely dissolved. 
SECTION: Lysis
9. The DNA solutions from both Falcon tubes are combined and the pooled DNA solution can be stored in 2-5 ml microtubes.
2. A sample of homogenized soil weighing no more than 5 grams was placed in a 50 ml Falcon tube and a CTAB solution (2% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES-acid, pH 5.3) was added to a final volume of 40 ml.  

SECTION: Lysis
3. Stir very vigorously on a stirrer and incubate overnight at 65°С.
TITLE: DeepSlice Automated Alignment
AUTHORS: Michael Henderson

[DESCRIPTION]
This protocol explains how to use DeepSlice in the Quint Workflow

[BEFORE_START]
DeepSlice is a deep neural network that automatically aligns mouse histology images through the Allen Brain Atlas coordinate framework. Alignments are viewable and refinable in QuickNII and set up sections to give a good starting point near and around each section’s correct plane. DeepSlice’s alignment is not completely accurate and further fine tuning in QuickNII is necessary. This is a newly developed tool and not imperative for the workflow but helps speed the process of registration and substitutes the FileBuilder step.

[STEPS]
SECTION: DeepSlice Automated Alignment
1. Open DeepSlice in your web browser.

 

SECTION: DeepSlice Automated Alignment
2. Select Choose Files.
a. Upload all images for registration from the QVN folder
SECTION: DeepSlice Automated Alignment
3. Ensure Mouse is selected for species, and Model Ensemble is checked (uses two DeepSlice versions to optimize alignment ).
a. Avoid checking Angle Integration (this aligns all your brain sections to the same angle, which is inaccurate when blocks have different cutting angles) 
b. Avoid checking Using Cutting Index (this suggests your sections numbers (_s###) correspond to serial section numbers spaced equally apart)  
c. Optional to allow DeepSlice to use your data to improve the neural network and its predictive accuracy.
SECTION: DeepSlice Automated Alignment
4. Select Submit.
SECTION: DeepSlice Automated Alignment
5. After all sections/slices are processed, press Download XML.
SECTION: DeepSlice Automated Alignment
6. Insert the downloaded XML (titled by default ‘results.XML’) into your QVN folder with all your images for registration.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>One-step metabarcoding PCR. Used to amplify either mitochondrial 12S or cytochrome B regions from environmental DNA sampled from freshwater to identify fish species. PCR product contains the sequencing primers ready for sequencing on the Illumina MiSeq (custom oligos required for sequencing).</p>

[GUIDELINES]
<p>References to primer sequences and their use with this method:</p>
<p> </p>
<p>Kocher, Thomas D., et al. "Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers." <em>Proceedings of the National Academy of Sciences</em> 86.16 (1989): 6196-6200.</p>
<div class="gs_citr" tabindex="0">
<div class="gs_citr" tabindex="0"> </div>
<div class="gs_citr" tabindex="0">Riaz, Tiayyba, et al. "ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis." <em>Nucleic Acids Research</em> (2011): gkr732.</div>
<div class="gs_citr" tabindex="0"> </div>
<div class="gs_citr" tabindex="0">
<div class="gs_citr" tabindex="0">Kelly, Ryan P., et al. "Using environmental DNA to census marine fishes in a large mesocosm." <em>PloS one</em> 9.1 (2014): e86175.</div>
</div>
</div>
<div class="gs_citr" tabindex="0">
<div class="gs_citr" tabindex="0">Hänfling, Bernd, et al. "Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods." <em>Molecular ecology</em> (2016).</div>
</div>

[STEPS]
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TITLE: Media for culturing cells
AUTHORS: Rebecca Berrens

[DESCRIPTION]
common ES cell media for mouse embryonic stem cells

[STEPS]
SECTION: ES cell media
1.  ES Medium
     Reagent      Volume      Supplier      Cat.number        DMEM   without sodium pyruvate*      500   ml      Invitrogen      41965-039        Fetal   calf serum ** (13% final)      75   ml      GlobePharm               Non-essential   amino acids, 100x      5   ml      Invitrogen      11140-035        L-glutamine,   200mM, (100x)      5   ml      Invitrogen      25030-024        b-mercaptoethanol, 50 mM      0.6   ml      Invitrogen      31350-010        Penicillin/Streptomycin      5   ml      Invitrogen      15140-122        LIF   (ESGRO) (now we use home-made LIF-conditioned medium)      1ml   (check current titer and add accordingly)                 

 
 
SECTION: EC10 media
2. Medium for differentiated ES cells and most fibroblast cultures (EC10)

 
 
     Reagent      Volume      Supplier      Cat.number        DMEM   without sodium pyruvate*      500   ml      Invitrogen      41965-039        Fetal   calf serum**      50   ml      GlobePharm               Non-essential   amino acids, 100x (optional)      5   ml      Invitrogen      11140-035        L-glutamine,   200mM, (100x)      5   ml      Invitrogen      25030-024        b-mercaptoethanol, 50 mM      0.5   ml      Invitrogen      31350-010        Penicillin/Streptomycin      5   ml      Invitrogen      15140-122
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol decribes how to make 1 L of Pseudomonas isolation agar for the culturing of Pseudomonas bacterial species on plates.

The use of a genus specific isolation media such as Pseudomonas isolation media reduces the risk of plate contamination with a diiferent bacterial species when trying to isolate a particular bacterial strain.

[GUIDELINES]
Once Pseudomonas isolation media has been autoclaved, all work should be carried out in a laminar flow hood to prevent contamination.

1 L of Pseudomonas isolation agar makes ~20 plates. 
At 4 °C unused plates can be stored for around 2 weeks prior to use.
Once used, plates containing bacterial colonies can be stored in air-tight container/bag at 4 °C for a maximum of 2 weeks.
Prior to disposal, plates containing bacterial colonies must be wrapped in Parafilm and autoclaved.

[STEPS]
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TITLE: Copy of   ELISA for quantification of IL-7 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Th9, Th2, Th17, mast cells, NKT cells, and regulatory T cells produce interleukin-9. It enhances T-cell survival, mast cell activation and synergy with erythropoietin.</span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-9 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-9 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-9 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-9 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-9 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-9 standard dilutions and the human IL-9 sample concentration determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol on how to publish your protocols on protocol.io. </p>

[BEFORE_START]
<p>Create a protocol. A tutorial on how to enter new protocols can be found <a href="https://www.youtube.com/watch?v=WVLu5hoVs7w" target="_blank" rel="noopener noreferrer">here</a>. </p>

[GUIDELINES]
<p>Make sure to double check that the protocol is ready for publication. Once published you can not edit or remove the protocol. However, you can always create a new version.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for LiAc <em>S. cerevisiae</em> competent cell preparation and transformation, from Aalto University Molecular Biotechnology group.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in "Isolation of cyanophages by plaque assays"

[GUIDELINES]
For example, purified agar or agarose (1% w/v) is added to your media of choice and autoclaved. This will provide a support base for the top agar/agarose overlay as well as nutrients for the host cells. For best results, use plates within 1 week of pouring.<br /><br /> Tip: plates can be fast-tracked: dry plates at 37°C; leave lids slightly ajar; monitor closely to prevent over drying.<br /><br /><em>Considerations:</em> Depending on the composition of the media used, the addition of solidification agents (in particular the combination of high salinity seawater-based media and common agar such as Bacto Agar) can often result in the formation of precipitates when autoclaved together. These flocks can sometimes interfere with interpretation of the plaque assay. Moreover, impurities in common agar can negatively affect the growth of the host cells. Here are some suggestions on how to reduce the formation of these precipitates. Some testing may be required to determine the best combination to use for your particular situation. <br /><br />a. Do not use common agar; rule of thumb the whiter the agar, the cleaner it is. <br />b. Use commercially available purified agar or agarose; or clean common agar using a washing procedure such as the one outlined in Waterbury and Willey (1989). <br />c. Reduce the salinity of seawater media with purified water; e.g., to 20-25 psu. <br />d. Add purified agar or agarose to autoclaved media aseptically and then melt the agar/agarose in the microwave (bring to a short boil 2-3 times to completely dissolve the agar/agarose). <br />e. For cells that will grow in artificial media, prepare media and gelling agent at 2x concentration and autoclave separately. When cooled to ca. 60°C, gently mix the gelling agent into the media and dispense immediately. <br />f. In the case of artificial media, add agar/agarose to filter-sterilized media and melt the gelling agent in the microwave

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TITLE: Vegan Latkes with JUST Egg
AUTHORS: Lenny Teytelman, Hannah Gershik, JUST

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recipe is a traditional Eastern-European Jewish latkes (potato pancakes). </div></div>

[STEPS]
?. [Grate potatoes and onion]
Peel and wash .
[russet potatoes]
?. [Mix all ingredients]
In a large mixing bowl mix the grated potatoes and onions with , , and .
[of salt]
[flour]
[JUST Egg]
?. [Grate potatoes and onion]
Cut up and grate the potatoes and .
[small yellow onion]
Either with a food processor fitted with a grater disc or just with a  box grater.
?. [Fry the fritters]
Cover bottom of a frying pan with oil and heat to medium-high.
?. [Grate potatoes and onion]
Use a spoon to remove the water.Put the spoon onto the surface of the batter, pressing it down a little bit. Let the liquid seep in.
?. [Fry the fritters]
Fry for 3-5 minutes until crisp.
?. [Fry the fritters]
Flip, add more oil, and fry another 3-5 minutes.
?. [Fry the fritters]
Line a plate with paper towels.
?. [Fry the fritters]
Once the oil is hot, roll ¼ cup of the fritter mixture into a ball and place itinto the oil using a fish spatula or slotted spoon.
If little kids are helping fry, be careful with dropping the batter onto the skillet as the oil can splash.
?. [Fry the fritters]
Transfer to the paper towel-lined plate.
?. [Fry the fritters]
Serve warm.
Eat them with vegan sour cream.