eamag/protocols-with-steps huggingface dataset:
TITLE: A Case of Congenital Hereditary Metabolic Disease-urea Cycle Metabolic Disorder
AUTHORS: 181830691 , sdadf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Congenital metabolic diseases are caused by the lack or abnormality of a certain enzyme or its cofactors, which can cause accumulation or lack of specific metabolites. The best outcome for children with inborn errors of metabolism (IEM) depends on recognizing the symptoms and signs of metabolic diseases, promptly assessing them, and transferring them to a medical center familiar with the evaluation and treatment of related diseases. Delayed diagnosis may lead to Acute metabolic decompensation, progressive nerve damage, or death.</div></div>

[STEPS]
TITLE: Measuring the amuont of bacteria in a soil sample
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Colony Forming Units (c.f.u) is a unit that is used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. It also depends on their ability to multiply under controlled conditions. In the paper published by </span><span style = "font-style:italic;">El-Hassan and Gowen, 2006</span><span>, they analyzed various formulations of </span><span style = "font-style:italic;">Bacillus subtilis </span><span>by counting the CFU of B. subtilis present in every formulated product.   </span></div></div>

[STEPS]
?. [CFU determination]
Colony Forming Units (CFU) can be determined by estimating the OD of spore suspension using a tube-reading spectrophotometer adjusted at 1.978 [corresponding to 8.5 · 1010 CFU/ml] at 600nm absorbance wavelength
?. [CFU determination]
The formulation will be placed on sterile aluminum foil in pans and air-dried for  with occasional stirring in a laminar airflow cabinet.
?. [CFU determination]
Dried formulations (35% moisture content) of B. mycoides will be passed through a 250μm mesh sieve to attain the desired particle size.
?. [CFU determination]
Pack in sterilized polypropylene bags, seal and store at room temperature prior to use.
?. [CFU determination]
Count CFUs to estimate the number of viable propagules of B. mycoides using the standard dilution platin method described in step 6.
?. 
?. [Performing standard dilution platin ]
?. [Performing standard dilution platin ]
mvsedfv
?. [Standard dilution method]
Take three   aliquots of the dried powder and place in   sterile PBST solution (this will include PBS +  Tween 20). Stir magnetically at high speed for . Now dilute this suspension with approximately and take   of this suspension and plate on Nutrient Agar (NA) media.
1 g
99 mL
0.2 mL
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The Qiagen QIAquick Gel Extraction kit (catalog #28704 and 28706) are for extraction of DNA fragments (70 bp – 10 kb) from standard or low-melt agarose gels in TAE buffer (Tris·acetate/EDTA) or TBE buffer (Tris·borate/ EDTA) and DNA cleanup from enzymatic reactions. The kit is suitable for purification of up to 10μg of DNA (70bp to 10kb).

[BEFORE_START]
<strong>Add ethanol (96–100%) to Buffer PE before use</strong> (see bottle label for volume).<br /><strong>All centrifugation steps are carried out at 17,900 x g (13,000 rpm)</strong> in a conventional tabletop microcentrifuge at room temperature (15–25°C).<br /><strong>Add 1:250 volume pH Indicator I to Buffer PB</strong> (i.e., add 120 μl pH Indicator I to 30 ml Buffer PB or add 600 μl pH Indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH Indicator I indicates a pH of ≤ 7.5. Add pH Indicator I to entire buffer contents. Do not add pH Indicator I to buffer aliquots. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH Indicator I.<br /><strong>Isopropanol (100%) and a heating block or water bath at 50°C are required</strong>

[STEPS]
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TITLE: Human Blood Sample Collection Protocol --University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Collection protocol obtained from the attached BioNet Specimen Procurement Agreement provided by the UMN CTSI Biorepository and Laboratory Services (BLS).

  


[STEPS]
SECTION: Preparation
1. Patient Identification: As soon as a patient is scheduled, the research team will email bionet@umn.edu a completed Specimen Procurement Request Form.
SECTION: Preparation
2. Patient Consent: Researcher consents. The original signed consent form will be placed in the patient chart and scanned into Epic. BioNet will verify consent in Epic. If the consent is not scanned in EPIC before the procedure, the researcher will provide BioNet a copy direct (e-mail or hard copy).
SECTION: Preparation
3. Collection Supplies: All supplies will be delivered to the BioNet office at least one day before the procedure and appropriately labeled with researcher name and CTSI Project #.

Collection Kit Components:
 PBS (Study team)
 Media: DMEM + antibiotics (Study team)
 Media tubes (x3 per collection) (Study team)
 10% Formalin (Bionet)
 FFPE cassette x 5 (Bionet)
 Specimen container (Bionet)
 OCT compound (Bionet)
 OCT mold x 4 (Bionet)
 OCT quick freeze box (Bionet)
 Liquid nitrogen vapor (Bionet)
 Cryovials x 3 (Bionet)
 Collection kit containing 10 ml EDTA blood tubes x 2 (Bionet)
SECTION: Procurement and Processing of Blood Samples
4. Study team will set up blood processing instructions with TTL.
Study team will place order for blood in EPIC. 
Study team will provide preop staff with blood tube collection kits. 2 x 10 ml EDTA blood tubes will be in a bag labeled with BLS pager number for pick up contact.
Blood tubes will be held at room temperature until delivery to TTL or pick up by study team.
BLS will pick up blood tubes and submit to TTL for processing. If filled blood tubes are returned to BLS past TTL’s 2pm processing deadline, study team will be notified to pick up blood tubes and process through their lab.
TITLE: PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans
AUTHORS: Coline Royaux

[DESCRIPTION]
This PCR protocol has been used to amplify the Cytochrome Oxydase I gene of neo-caledonian genera Boeckella, Lynceus, Eulimnadia and Streptocephalus with several primers.

[STEPS]
1. Prepare the mix for the PCR depending on the quantity of DNA samples you want to amplify
 

 



1.1. For each well, mix 12.44 µL, 2 µL, 1 µL, 1 µL, 0.8 µL and 0.12 µL. This mix is hereafter named "intermediary mix".
 


1.2. Add your primers to the mix, 0.32 µL 10 picomolar (pM) and 0.32 µL 10 picomolar (pM)  for each well. This mix is hereafter named "final reagent mix".
 

 



2. In each well, deposit 18 µL and 2 µL unless for the negative control.
For the positive control, use the DNA of the species on which the primer has been designed.

3. Close the PCR plate with a heated aluminium foil and centrifuge it enough to make sure the reagents are all in the bottom of the well.
4. To amplify targeted DNA sequences, place your plate in the thermocycler, set and launch the cycle as described in the following sub-steps
4.1. 5 min at 94 °C 

4.2. 40 cycles of the following steps :
30 s at 94 °C 
30 s at 50 °C 
1 min at 72 °C 

4.3. 8 min at 72 °C 

4.4. Infinite timing at 20 °C so the samples are well preserved and you have some time to pick it up

5. To know if your amplifications worked properly, you now have to proceed to a gel electrophoresis of your PCR products
5.1. To prepare the gel,
 

 mix 0.8 mg and 40 mL, heat the preparation in the microwave for a few moments until it is homogeneous (you can agitate the preparation). Wait until the preparation is cold enough to touch its container and add 0.8 µL.
5.2. Pour the preparation in a mold and let the gel polymerize with a bar to form wells to deposit your PCR products
5.3. Deposit the gel in the electrophoresis tank
5.4. Fill each well with 2.5 µL and 0.5 µL , don't forget to save a well to deposit 1.2 µL 

5.5. Start electrophoresis in TAE buffer for 15 min at 200 Volts

5.6. Look at your gel under Ultra Violet light to see the results
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>T regulatory cells (also known as Tregs or Regulatory T cells) are essential cells in<br />the immune system that suppress immune responses of other cells, designed to<br />limit excessive reactions and prevent autoimmunity. Tregs are characterized by the<br />expression of CD4, CD25, and Foxp3, while lacking CD127. CD4+Foxp3+ regulatory<br />T cells have been referred to as “naturally-occurring” regulatory T cells to distinguish<br />them from “suppressor” T cell populations that are generated in vitro. While other<br />variants of suppressive T cells do exist, such as CD8 suppressor cells, Th3 and Tr-1 cells,<br />Tregs are classically defined as CD4+CD25+Foxp3+ cells. Here, we provide a protocol<br />for immunofluorescent staining of FOXP3 in frozen sections, adapted from a protocol<br />from Dr. Matthias Hardtke-Wolenski, Medizinische Hochschule Hannover. This protocol<br />has been successfully used on mouse spleen, liver, and skin sections.</p>

[GUIDELINES]
<p><strong>Antibody Dilution Buffer</strong>:</p>
<p><br />•1%BSA (blocking &amp; stabilizer)<br />•0.1% cold fish skin gelatin (blocking)<br />•0.5% Triton X-100 (penetration enhancer)<br />•0.05% sodium azide (preservative)in TBS</p>
<p> </p>
<p>Mix well and store at 4ºC.</p>

[STEPS]
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TITLE: cermep-bids-retro
AUTHORS: lucie.chalet

[DESCRIPTION]
cermep-bids-retro enables the formatting of retrospective PET and MRI data to BIDS standards from DICOM databases. When dealing with pre-clinical imaging modalities, numerous metadata are overlooked during acquisitions making it difficult to automatically format following BIDS guidelines. This python-based project is a flexible method for BIDS formatting with input csv files to compensate for lacking DICOM tags. And advanced data quality assessment.

[STEPS]
SECTION: cermep-bids-retro installation
1. Clone repository
In GitBash:
 

SECTION: cermep-bids-retro installation
2. Create conda environment

 

SECTION: Fill-in input files
3. This step is key to a functional method. It requires a good knowledge
of the available tags and fields in the DICOM data and one can always refer
to the BIDS specifications to define all required tags for their modality.
Substeps below provide description and example for each column of the 3 input files. 
SECTION: Fill-in input files
3.1.   

SECTION: Fill-in input files
3.2. This table must be filled in simultaneously with seq_overview.csv
 
SECTION: Fill-in input files
3.3. This table is necessary for formatting of PET data, otherwise optional
  

SECTION: Run formatting task
4. Replace argument values and run task from command line:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time PCR for the detection of Coxiella burnetii DNA targeting the IS1111a gene. The assay is based on a published method by Banazis et al 2010,  using a different PCR kit. The oligonucleitide sequences have not been modified however the concentration have been optimised. This method is used for testing DNA from human blood and tissue samples.</p>

[GUIDELINES]
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
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TITLE: ROS staining for Arabidopsis (green fluorescent stain)
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is adapted from two publications describing fluorescent staining for ROS - </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Shin et al., 2005</span></a><span> and </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Orman-Ligeza et al. 2016</span></a><span> (citing Shin)</span></div></div>

[STEPS]
?. Transfer the seedlings from agar plates into nutrient solution that is the same as agar plate composition (but without Dashin agar). You can use 24/12/6 well-plates - this will depend on your final seedlings size
?. Wash the seedlings with medium without DCF-DA
?. Sterilize the seedlings, germinate and transfer to for e.g. salt stress medium as described in the "Quantification of salt-induced changes in Root System Architecture in Arabidopsis" protocol - dx.doi.org/10.17504/protocols.io.zkqf4vw
?. After transferring the seedlings to liquid medium add DCF-DA so that the final concentration of the DCF-DA in the incubation medium is 50 uM
?. Wrap the well plate in aluminium foil (DCF-DA is light sensitive) and let the samples incubate for 30 min with gentle shaking.
?. Image the stain using fluorescence microscopy: - 460–500 nm bandpass excitation- 510– 560 nm bandpass emission
TITLE: Zebrafish Embryo Dissociation for MACS
AUTHORS: Sam Wattrus

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method of zebrafish embryo dissociation and microbead binding for magnetic enrichment by MACS. </div></div>

[STEPS]
?. Prepare enzyme mix by adding 200ul Enzyme H, 100ul Enzyme R, and 25ul Enzyme A to 4.7mL DMEM on ice.
?. Tricaine and transfer embryos in minimal E3 to plate lid. Remove excess E3 (can be done effectively by blocking embryos with razor blade and wicking up water behind the blade with a Kimwipe). Chop embryos in plate lid. Make sure blade is in full contact with bottom surface, rotating the plate lid periodically.
?. Recover in 1.5mL enzyme mix to Eppendorf tube.
?. Vortex and incubate for 15-20 minutes at 37C in thermomixer. Vortex and pipet up and down vigorously every ~5 minutes until sample is dissociated.
?. Add FBS to 10% to quench (~150ul).
?. Filter through 40um blue cap FACS tubes on ice.
?. Transfer to fresh Eppendorf tubes and spin down 4 minutes @ 800rpm (tabletop centrifuge – aprox 60xg).
?. Carefully remove and discard supernatant. Resuspend cells in 1mL cold PBS. Take 1ul and add to 99ul Trypan blue in an Eppendorf to count a 1:100 dilution.
?. Spin down in tabletop centrifuge for 5 minutes at 300xg.
?. While cells are spinning down, mix 1:100 dilution and use 10ul to count total cell number on a hemocytometer.
?. Remove and discard supernatant, resuspend in 60ul MACS Buffer per 107 total cells. Add 20ul Microbeads per 107 total cells.
?. Mix well and incubate for 15 minutes @ 4C protected from light.
?. Wash by adding 1-2 mL MACS Buffer and centrifuge 5 minutes at 300xg, aspirate supernatant completely.
?. Resuspend up to 108 cells in 500ul of MACS Buffer.
?. Proceed to magnetic separation.These cells are now compatible for use with either the AutoMACS Pro separator or manual column separation.
?. If proceeding to single cell RNA sequencing after magnetic enrichment, cells should be washed with PBS/0.5% BSA, spun down and resuspended before counting on a hemocytometer. EDTA in the MACS buffer can inhibit reverse transcription, so this should be removed.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Tips</strong>:</p>
<p><strong>High background</strong></p>
<p>1. Transfer buffers may have become contaminated. Contamination can be transferred to the blots from electrophoresis and related equipment used in blot preparation.</p>
<p>2. Post-antibody washes may not have been performed for a sufficient period of time or were not performed in a high enough volume.</p>
<p>3. The blocking and incubation agents used were not freshly prepared or were too dilute.</p>
<p> </p>
<p><strong>No signal or poor signal</strong></p>
<p>1. Transfer efficiency may have been poor. Check protein transfer by staining the gel and/or membrane.</p>
<p>2. Incorrect storage of antibodies or ECL western blotting detection reagents may result in a loss of signal.</p>
<p>3. Insufficient protein may have been loaded on the gel. Depending on the location of the target protein, membrane or nuclear preparations may be required (instead of whole cell lysates).</p>
<p>4. Film exposure time may have been too short.</p>
<p> </p>
<p><strong>Solutions and Reagents:</strong></p>
<p> </p>
<p><strong>1X Cell Lysis Buffer: </strong></p>
<p>20 mM Tris-HCl, pH 7.5</p>
<p>150 mM NaCl</p>
<p>1% NP-40</p>
<p>2 mM EDTA</p>
<p>1 µg/ml leupeptin</p>
<p>1 µg/ml aprotinin</p>
<p>1 mM Na<sub>3</sub>PO<sub>4</sub></p>
<p>1 mM PMSF</p>
<p>5 mM NaF</p>
<p>3 mM Na<sub>4</sub>P<sub>2</sub>O<sub>4</sub></p>
<p> </p>
<p><strong>5X SDS Sample Buffer:</strong></p>
<p>312.5 mM Tris-HCl (pH 6.8)</p>
<p>10% SDS (w/v)</p>
<p>250 mM DTT</p>
<p>50% Glycerol</p>
<p>0.05% Bromophenol Blue (w/v)</p>
<p>Use at 1X</p>
<p> </p>
<p><strong>10X SDS Running Buffer</strong></p>
<p>Dissolve 144 g of Glycine, 30 g of Tris base and 10 g SDS in 800 ml of distilled H2O.</p>
<p>Add distilled H2O to 1 liter</p>
<p>Use at 1X</p>
<p> </p>
<p><strong>Transfer Buffer:</strong></p>
<p>3.0 g Tris base</p>
<p>14.4 g Glycine</p>
<p>200 ml Methanol</p>
<p>Add distilled water to 1.0 L</p>
<p> </p>
<p><strong>10X TBS-T (Tris-buffered saline containing Tween-20):</strong></p>
<p>Dissolve 80 g of NaCl, 2 g of KCl, 30 g of Tris base and 10 ml Tween-20 in 800 ml of distilled H<sub>2</sub>O.</p>
<p>Adjust the pH to 7.4 with HCl. Add distilled H2O to 1 liter.</p>
<p>Use at 1X (containing 0.1% Tween-20).</p>
<p> </p>
<p><strong>Blocking Buffer:</strong></p>
<p>1X TBS-T with 5% nonfat dry milk</p>
<p> </p>
<p><strong>Wash Buffer:</strong></p>
<p>1X TBS-T</p>
<p> </p>
<p><strong>Direct-Blot™ Antibody Dilution Buffer:</strong></p>
<p>1X TBS-T with 5% nonfat dry milk</p>
<p> </p>
<p>**If phosphorylation-specific antibodies are used, the membrane blocking buffer and antibody dilution buffer should not contain milk.</p>
<p> </p>
<p><strong>Alternate Blocking Buffer:</strong></p>
<p>1X TBS-T with 4% Bovine Serum Albumin (BSA)</p>
<p> </p>
<p><strong>Alternate Direct-Blot™ Antibody Dilution Buffer:</strong></p>
<p>1X TBS-T with 4% Bovine Serum Albumin (BSA)</p>
<p> </p>
<p><strong>Blotting Membrane:</strong></p>
<p>Nitrocellulose or PVDF membrane</p>

[STEPS]
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TITLE: LRRK2 thermal shift assay
AUTHORS: Verena Dederer, Deep Chatterjee, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
Thermal shift assay or differential scanning fluorimetry analyzes the effect of small molecules on the thermostability of a protein by gradual heat denaturation and monitoring absorption of the fluorescent dye SYPR Orange at 488 nm.


[STEPS]
SECTION: Fluorescent-based thermal shift assay
1. Prepare 4 µM master mix of protein in buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol) and add 1:1000 dilution of SYPR Orange.
SECTION: Fluorescent-based thermal shift assay
2. Aliquot 20 µL of the master mix into a white 96 well plate.
      
SECTION: Fluorescent-based thermal shift assay
3. Add DMSO or small molecule binder with a final concentration of 10 µM.

SECTION: Fluorescent-based thermal shift assay
4. Seal plate, mix well and centrifuge 30 sec at 500xg.
SECTION: Fluorescent-based thermal shift assay
5. Place plate into MX3005P real-time PCR instrument.
SECTION: Fluorescent-based thermal shift assay
6. Measure fluorescence with excitation and emission filters set to 465 and 590 nm while gradually increase temperature 3K/min during 71 cycles from 25 to 95°C.
TITLE: Proteomic Sample Preparation
AUTHORS: Shiyi Wang

[DESCRIPTION]
Proteomic Sample Preparation

[STEPS]
1. **Sample Storage and Preparation** - Received 12 samples (3 of each WT CYT, GS CYT, WT EZR, and GS EZR) and kept at -80°C until processing. - Spike samples with undigested bovine casein at a total of either 1 or 2 pmol as an internal quality control standard.
2. **Reduction and Alkylation** - Reduce samples for 15 minutes at 80°C. - Alkylate with 20 mM iodoacetamide for 30 minutes at room temperature.
3. **Protein Trapping** - Supplement samples with a final concentration of 1.2% phosphoric acid and 636 μL of S-Trap (Protifi) binding buffer (90% MeOH/100 mM TEAB). - Trap proteins on the S-Trap micro cartridge.
4. **Digestion** - Digest trapped proteins using 20 ng/μL sequencing grade trypsin (Promega) for 1 hour at 47°C.
5. **Elution** - Elute digested proteins using the following solutions in sequence: - 50 mM TEAB - 0.2% FA - 50% ACN/0.2% FA
6. **Lyophilization** - Lyophilize all samples to dryness.
7. **Resuspension** - Resuspend samples in 12 μL of 1% TFA/2% acetonitrile with 12.5 fmol/μL of yeast ADH.
8. **Study Pool QC (SPQC) Creation** - Create a study pool QC (SPQC) by combining equal volumes of each sample.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>osmFISH is a cyclic single molecule fluorescent<em> in situ</em> hybridization protocol used to quantify the expression level of specific transcripts in tissue sections by direct labeling of individual RNA molecules. The number of transcripts quantified in each round correspond the numbers of fluorophores available in the microscope setup. In order to quantify a large number of genes, osmFISH provides a method to remove the probes/labelling (stripping) from their targets and get the tissue ready for the next round of labeling - imaging - stripping.</p>
<p> </p>
<p>Even though the osmFISH protocol has been developed to perform multiple smFISH rounds, it can also be used as a quick and simple method for one-round smFISH. Furthermore, the protocol can be further extended to include more complex encoding/barcoding shemes that can be used to resolve a larger number of targets. </p>

[BEFORE_START]
<p>Please reed our considerations about the imaging and hybridization chamber setup on our <a href="http://linnarssonlab.org/osmFISH/gettingstarted/" target="_blank" rel="noopener noreferrer">website</a>.</p>
<p> </p>
<p>Prepare the Hybridization mix. We tipically make 10-40ml, aliquot and freeze at -20°C untill use. </p>
<p> </p>
<p>Aliquot the formamide in 200ml aliquots and freeze at -20°C. At the start of each week we thaw wat we need that week.</p>

[GUIDELINES]
<p>The volume of the wash steps are determined by the volume of the flow cell. At least replace the volume once. The wash volume we use is double that of the flow cell. So 2 x 500µl = 1ml per wash, in our case.</p>
<p> </p>
<p><a href="https://www.biosearchtech.com/support/education/stellaris-rna-fish" target="_blank" rel="noopener noreferrer">Stellaris RNA FISH probes</a> were orderd from Biosearch Tecnologies</p>

[STEPS]
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TITLE: Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation.

Collection Overview 
Preparing MEF-cultured hPSCs for nucleofection
Preparing Feeder-free hPSCs for nucleofection
Preparing plasmids for nucleofection
Preparing mRNA for nucleofection
In vitro assembling of RNP for nucleofection
Nucleofection of hPSCs
Electroporation of hPSCs
    A. Preparation of MEFs-cultured hPSCs for electroporation
    B. Preparing plasmids for electroporation
    C. Electroporation
FACS-based enrichment of transfected cells 
    A. Preparation of samples for FACS sorting
    B. After FACS

General notes
Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

[STEPS]