eamag/protocols-with-steps huggingface dataset:
TITLE: Cytochrome C Assay
AUTHORS: Elizabeth Fozo

[STEPS]
?. [Steps]
Start ON cultures of a strain of interest.
?. [Steps]
The next day, measure 100 mL BHI into each flask to be used.
?. [Steps]
Measure OD600 of each overnight and calculate how much of your overnight culture you need for OD600~ 0.01in 100mL.
TKO – 20 min – WT – 10 min – DKO
?. [Steps]
Add supplement if doing Long Term exposure or place BHI + 0.01 cells directly into 37*C for a later spike-in
?. [Steps]
Harvest cells at 0.3 if doing Long Term or 0.225-0.25 if doing Spike
Check OD after 2.5 hours.
?. [Steps]
Obtain OD600 values for each of your cultures and then calculate how much of your culture is needed to reach an OD600 of 7 in 3 mL
Formula: (7-OD600)(3mL) = (measured log phase-OD600)(X mL)Will need around 60-80mL to achieve this concentration
?. [Steps]
Pipet half the total volume needed into a 50mL conical and centrifuge for~10 minutes at 3500 RPM. Pour off supernatant.  Add the other half of the cell volume.Centrifuge again.
?. [Steps]
Wash the cells with 10 mL of 20mM MOPS buffer*(stock is 1M) by centrifugation for 5 min at 3500 RPM.  20mM MOPS buffer is made in H2O
C1V1=C2V2 formula to figure out how much stock to make 20mM.*MOPS-3-(N-Morpholino)propane sulfonic acid resuspend in H2O– autoclave.
?. [Steps]
Repeat the wash
?. [Steps]
Resuspend the pellet in 3 mL of 20mM MOPS buffer
?. [Steps]
Add 1mg/mL of Cytochrome C (stock is 10mg/mL in H2O)- vortex
C1V1 = C2V2
?. [Steps]
Incubate for 10 minutes
?. [Steps]
Centrifuge for 10 minutes at 3500 RPM
?. [Steps]
Add 2 mL of the supernatant to 4mL plastic cuvettes
?. [Steps]
While cytochrome cells are spinning, make standards. To 3 mL of 20mM MOPS buffer add:0.1 = 30uL of cytochrome0.2 = 60uL of cytochrome0.3 = 90uL0.4 = 120uL
?. [Steps]
Use the Cytochrome program (“CYTOCHROME”) on the Fozo Lab Spec = OD530
Standards listed above cover a range of predetermined mg/mL results, if your results fall outside of this range, you may need to adjust your standards accordingly.
TITLE: Enzyme linked immunosorbent assay for investigating the immunoglobulin-binding bacterial protein (IBBP) to avian egg yolk antibodies.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. This ELISA is used to study the interaction of proteins A, L and LA with different avian IgY preparations.
?. The 96 well microtitre plate is  coated overnight at 4°C with 1 µl/mg per well of unlabelled SpL, SpA or SpLA in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. 50 µl of different  IgY preparations (1 mg/ml) are added and incubated for 1h at room temperature and washed.
?. Then 50 µl of rabbit anti-chicken IgG-HRP conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and incubated for 1h at RT. The plate is washed 4X with PBS-Tween.
?. 50 µl of 4 mg/ml o-phenylenediamine solution (OPD) is added and the plates are incubated 15 minutes at RT.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour.
?. The plate is read in a microplate reader at 492 nm.
?. A cut-off point is calculated.
TITLE: 10 Affinity Chromatography
AUTHORS: TJUSLS China

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

[STEPS]
?. Lysis of the bacteria.1. Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 mL pipet or whatever means    necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.2. Smash the bacteria.3. Centrifuge at 18,000 rpm in a big rotor for 40 minutes at 4°C. Save the pellet and the supernatent.
10 ml
4 °C
?. IMAC(Immobilized metal ion affinity chromatography)Procedure1. Remove the Ni column from the 4℃ refrigerator, which contains 20% alcohol. Wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.2. Add the protein solution to the column, let it flow naturally and bind to the column. Repeat until the medium turns gray ( usually twice ).3. Add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.4. Add Ni-Elution buffer several times. Check as above.5. Collect the eluted proteins for further operation. Recycle columns: Wash with 0.2mM EDTA , let stand for 10 min then drain. Wash with 6M Guanidine hydrochloride, let stand for 10 min then drain. Wash with ddH2O for three times. Fill up with NiSO4, place on a shaker overnight at 4℃.GST（Affinity chromatography of glutathione transferas）Procedure1. Remove the GST column from the 4℃ refrigerator. Wash the column with GST-binding buffer for 10 minutes to balance the GST column.2. Add the protein solution to the column, let it flow naturally and bind to the column.3. Add GST-Washing buffer several times and let it flow. Take 5μl of wash solution and test withCoomassie Brilliant Blue. Stop washing when it doesn’t turn blue.4. Add GST-Elution buffer several times. Check as above.5. Collect the eluted proteins for further operation. Recycle columns: Wash with GST-Elution buffer until Coomassie Brilliant Blue doesn’t turn blue when tested. Wash with 6M Guanidine hydrochloride , let stand for 10 min then drain. Wash with ddH2O for three times.
4 °C
5 µl
4 °C
4 °C
5 µl
TITLE:       ELISA for quantification of IL-40 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>IL-40 is produced in the bone marrow, fetal liver, and by activated B cells. IL-40 plays a vital role in the development of humoral immune responses. </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-40 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-40 present in the serum sample binds to antibodies adsorbed into 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-40 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-40 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) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-40 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-40 standard dilutions and the human IL-40 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Transfection of mammalian cell lines with plasmids and siRNAs
AUTHORS: Agnes Roczniak-Ferguson, Shawn M. Ferguson

[DESCRIPTION]
This protocol details the transfection of mammalian cell lines with plasmids and siRNAs.

[STEPS]
SECTION: Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
1. On the day before transfection, plate 100,000 HeLa cells per well in a 6 well dish. For other cell lines, the number of cells will need to be optimized to achieve 50-75% confluency on the day of transfection.
SECTION: Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
2. Warm Optimem (Gibco) and transfection reagent to Room temperature
SECTION: Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
4. Incubate 20 min at Room temperature.

SECTION: Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
5. Add dropwise to cells and mix gently. Return cells to incubator. Duration of transfection must be optimized for each plasmid and downstream application.
SECTION: RNAiMax transfection reagent
6. Warm Optimem (Gibco) and RNAiMax (Invitrogen) transfection reagent to Room temperature.

SECTION: RNAiMax transfection reagent
7. Combine 200 µL OPTiMEM, 5 µL RNAiMAX transfection reagent and 5 µL of 20 1534 siRNA and mix gently.

SECTION: RNAiMax transfection reagent
8. Incubate 20 min at Room temperature. 

SECTION: RNAiMax transfection reagent
9. Add transfection mix to 1.8 mL media containing 100,000 HeLa cells per well in 6-well dish and return to cell culture incubator for 24-72 hours (duration needs to be optimized for each target gene).

SECTION: Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
3. Add 200 µL Optimem + 6 µL transfection reagent + 2 µg plasmid DNA to a round bottom polystyrene tube and mix gently.
TITLE: Preparing mitochondrial samples for immunoblotting
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for preparation of mitochondrial samples for immunoblot analysis

[STEPS]
1. Thaw mitochondrial stocks on ice, and aliquot out the desired amount of mitochondria.
2. Centrifuge each aliquot for 10 min  10000 x g, 4 °C 

3. Carefully aspirate the supernatant from each sample. 
4. Add a volume of 1x SDS sample buffer (5% w/v SDS, 10% v/v glycerol, 100 mM DTT, 50 mM Tris-Cl pH 6.8) equal to the amount of mitochondria (in ug) to each sample. Eg. If each sample contains 20 µg of mitochondria, add20 µL of 1x SDS sample buffer.

5. Vortex samples for ~5 seconds to mix, and then boil at 99 °C with shaking at max speed for 10 min. 

6. Allow samples to cool to room temperature, quickly centrifuge to collect liquid to the bottom of the tube, and vortex for ~3 seconds to ensure the samples are homogenous. 
7. Samples can now be directly loaded onto an SDS-PAGE gel, or stored at -20 °C for later use.
TITLE: The  role of sphingolipids in the pathogenesis of psoriasis
AUTHORS: Mateusz Matwiejuk, Hanna Mysliwiec, Adrian Chabowski, Iwona Flisiak

[DESCRIPTION]
Psoriasis is complexed, chronic, immunologically mediated disease, which involves skin and joints. Psoriasis is commonly connected with numerous other diseases such as liver diseases, metabolic syndrome, impaired glucose tolerance, diabetes mellitus, atherosclerosis, hypertension, ischemic heart disease. Interestingly, comorbidities of psoriasis are attention-grabbing issue, additionally it can cause impairment of quality of life and may be associated with depressive disorders. Altered levels of ceramides in psoriatic skin, may lead to an anti-apoptotic and pro-proliferative states, consequently conducting to over-proliferation of keratinocytes and the development of skin lesions. Pathophysiology of psoriasis and its comorbidities is not fully understood yet. Sphingolipids, including ceramides, and their disturbed metabolism, may be the link between psoriasis and its comorbidities. Overall, the goal of this review was to discuss the role of sphingolipid disturbances in psoriasis and its comorbidities. PubMed was searched from February to the beginning of the May 2022. The systematic review included 73 eligible original articles. 

[STEPS]
1.
TITLE: H_QTof_PL_v1
AUTHORS: UTD Mass Spec Core, M, Fang Bian

[DESCRIPTION]
To extract plasma lipids for LCMS analysis.

[BEFORE_START]
Use LCMS grade water and solvents. 

[GUIDELINES]
Use glassware throughout this protocol. 

[STEPS]
SECTION: To extract plasma lipids for LCMS study
1. Transfer plasma 50 ul (regular pipette tips) into a 3 ml glass tube
SECTION: To extract plasma lipids for LCMS study
2.  Add 200 ul of LCMS grade-chloroform/methanol (2:1)
SECTION: To extract plasma lipids for LCMS study
3. Stand for 5 min at room temperature.
SECTION: To extract plasma lipids for LCMS study
4. Vortex for 1 min
SECTION: To extract plasma lipids for LCMS study
6. Repeat steps 3-5
SECTION: To extract plasma lipids for LCMS study
8. Centrifuge 12,000 g, for 5 min at 4c
SECTION: To extract plasma lipids for LCMS study
7. Use glass pipette tips to collect the lower organic phase and transfer the tube in step 5.
SECTION: To extract plasma lipids for LCMS study
9. Evaporate to dryness 
SECTION: To extract plasma lipids for LCMS study
10. Cap the tube, store at -20oC
SECTION: To extract plasma lipids for LCMS study
11. Deliver the sample to mass spec core.
SECTION: To extract plasma lipids for LCMS study
5. Use glass pipette tips to collect the lower organic phase and transfer the liquid to a new glass tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a workflow for platereader measuremtns with V. natriegens.

[STEPS]
SECTION: Sample preparation
?. 
SECTION: Sample preparation
?. 
SECTION: Sample preparation
?. 
SECTION: Sample preparation
?. 
SECTION: Preculture
?. 
SECTION: Preculture
?. 
SECTION: Preculture
?. 
SECTION: Preculture
?. 
SECTION: Measurement
?.
TITLE: Infecting Cells with SeV or RSV in A549 or LLCMK2 cells
AUTHORS: Carolina Lopez

[DESCRIPTION]
Infection of SeV or RSV in A549 or LLCMK2 cells

[STEPS]
SECTION: Infecting Cells with SeV or RSV in A549 or LLCMK2 cells
1. Infecting cells with SeV or RSV in A549 or LLCMK2 cells

1. Prepare a virus dilution in infection media with the correct virus-specific media (see materials section for media composition).
Calculate the volume of virus needed (X) using the MOI formula: 
     (MOI*cell number)/Virus titer = X
The amount of infection media used to prepare the virus will depend on the size of the well or plate used - it is best to use the minimal volume needed to cover the well/plate to help ensure virus particle attachment to cells 
2. Remove TCM
3. Wash cells twice with 1X PBS
4. Add XµL of virus suspended in infection media to each well (calculated in step 1)
5. Incubate the plate for 1 hr @ 37°C, rocking the plate/flask every 15 minutes 
6. Remove the media 
7. Wash twice with 1X PBS
8. Add infection media to each well/flask 
For SeV infection: add SeV-Infection media, for SeV infection in LLCMK2 cells: add TRYPSIN –TPCK to the infection media for a final concentration of 2µg/mL TRYPSIN –TPCK
For RSV infection: add RSV-Infection media (2%FBS)
9. Place the plate/flask back in a 37ºC, 5% CO2 incubator
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We found many specific steps and conditions for gram-negative bacteria while working with DNeasy Blood &amp; Tissue Kit. These details are described in DNeasy Blood &amp; Tissue Handbook (https://www.qiagen.com/us/resources/resourcedetail?id=6b09dfb8-6319-464d-996c-79e8c7045a50&amp;lang=en). However we decided to describe this protocol step by step pointing out in some critical steps. This protocol was successfully applied while genomic DNA extraction from Shigella spp. and Salmonella spp. strains for whole-genome sequencing.</p>

[BEFORE_START]
<p>Add <strong> Ethanol 96%</strong> to reagents of QIAGEN kit as described by vendor </p>
<p> </p>
<p><strong>Prepare CSB solution (Cell suspension buffer)</strong> </p>
<p>1 M Tris-HCl (pH 8.0) - 10 ml</p>
<p>0.5 M EDTA (pH 8.0) - 20 ml </p>
<p>Add  deionized water to 1 Litre </p>
<p>Store at +4°C to +8°C </p>
<p> </p>
<p>Prepare <strong>Proteinase K solution (20 mg/ml)</strong> in deionized water</p>
<p>Store at -20°C</p>
<p> </p>

[STEPS]
?. 
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?. 
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TITLE: Automatic labeling tissue and cell of human skin
AUTHORS: kyu sang han, peihsun.wu

[DESCRIPTION]
This is first upload from TMC - Johns Hopkins

[STEPS]
SECTION: Tissue biopsy collection
1. After punch/excisional biopsy from a operating room (OR), place the skin tissue into a tissue container prefilled with buffered formalin for 12-24 hours at room temperature (RT)
SECTION: Tissue biopsy collection
2. Discard formalin, rinse with PBS, refill PBS, and leave the tissue in PBS for 1 minute
SECTION: Tissue biopsy collection
5. Put cassette with the tissue back into the tissue container filled with PBS
SECTION: Paraffin embedding
6. Dehydrate tissue by submerging cassettes into ethanol at increasing concentrations from 70% to 99%.
SECTION: Tissue biopsy collection
3. Take tissue out on a large Petri dish and measure the tissue size
SECTION: Tissue biopsy collection
4. Label a slotted cassette with a pencil with tissue ID and place the tissue in the cassette
SECTION: Paraffin embedding
7. Embed cassette paraffin at 60 deg C and store at room temperature
SECTION: Tissue sectioning
8. Section tissue and place on water bath to expand
SECTION: Tissue sectioning
9. Place expanded tissue section on a Superfrost Plus microscope slide
SECTION: Tissue scanning
10. Scan unstained microscope slide at 40X magnification using Hamamatsu nanozoomer S210
SECTION: Converting image format from ndpi to ome.tiff
11. Use openslide library in python along with pyvips library to load whole slide image file in proprietary format (ndpi) from Hamamatsu and save as ome.tiff along with metadata.
SECTION: Applying semantic segmentation model in MATLAB
12. Load the ome.tiff image using bioformat library for MATLAB and generate tiles to input into a deep learning model to semantically segment 11 different skin tissue compartments.
TITLE: Instructions of the Experiment - Contrasting effects of information sharing on common-pool resource extraction behavior: experimental findings
AUTHORS: Dimitri Dubois, Rouchier Juliette, Nguyen-Van Phu, Farolfi Stefano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Instructions of the experiment of article "Contrasting effects of information sharing on common-pool resource extraction behavior: experimental findings".</div></div>

[STEPS]
TITLE: The blueprint and materials for building an imaging platform
AUTHORS: Wei-Ping Chan

[DESCRIPTION]
Details could be found in the Supplementary Information of "A high-throughput multispectral imaging system for museum specimens"

[STEPS]
2. Materials
3. Other accessory designs that improve workflow
2.2. Imaging platform
Materials:
 
Tools:
 

1. Blueprint

   

3.1. Special clips for label removal and attachment
Hairdressing double prong pin (1.8") link
This special clip can remove the labels and keep the pin holes labels aligned. It saves time for reattaching labels back on to the specimen after imaging.
3.2. Magnetic bar for mounting pin with removed labels.
Heavy-duty magnetic tool holder link
This bars help keep the clips in 3.1 in order, so the labels can be reattached to the correct specimens.
2.1. Scales and standard references
Materials:

 
Tools:
TITLE: First strand cDNA synthesis (ThermoScientific RevertAid)
AUTHORS: Ben Kuipers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol is optimized to generate first-strand cDNA for use in (q)PCR</div></div>

[STEPS]
?. Add reaction components into sterile, nuclease-free tube on ice in the indicated order:  AB1Template RNA100 ng&nbsp; ( 1pg - 5 µg)2Oligo(dT)181 µl (100 pmol)3Water, nuclease-freeto 12 µl
AB1Template RNA100 ng&nbsp; ( 1pg - 5 µg)2Oligo(dT)181 µl (100 pmol)3Water, nuclease-freeto 12 µl
?. Optional: If the RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65 °C for 5min. Chill on ice, briefly centrifuge again and place on iceice.
?. AB15X RT Buffer4 µl2RiboLock RNase Inhibitor1 µl (20 U)3RevertAid RT (200 U/µL)2 µl (400 U)410 mM dNTP Mix1 µl5Total volume20 µl
Mix gently and centrifuge briefly.
AB15X RT Buffer4 µl2RiboLock RNase Inhibitor1 µl (20 U)3RevertAid RT (200 U/µL)2 µl (400 U)410 mM dNTP Mix1 µl5Total volume20 µl
?. AB15 min25 °C260 min42 °C (For GC-rich RNA, the reaction temperature can be increased to 45 °C)35 min70 °C
AB15 min25 °C260 min42 °C (For GC-rich RNA, the reaction temperature can be increased to 45 °C)35 min70 °C
?. The cDNA product is now ready for downstream applications
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
1 µl 10x CutSmart buffer, 1 µl T4 DNA ligase buffer, 0.25µl U6 plasmid (about 100ng), 1µl annealed oligos, 0.3 µl T4 DNA ligase, 0.3 µl BsmBI, 0.2µl PstI (optional), 0.2 µl SalI (optional), 5µl H2O

[STEPS]
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TITLE: Microplate Reader Workflow
AUTHORS: NUS iGEM

[STEPS]
?. Refresh overnight cell culture in LB media at
37 °C
?. Measure OD600 of cell culture for desired OD value
?. Load  of cell samples into three wells of a 96-well plate (triplicates)
200 µl
?. Induce appropriate volumes of chemical inducers (not exceeding ) in each well
6 µl
?. Include LB media as blanks
?. Load the plate (with lid) into microplate reader
?. Start protocol to run continuous measurement overnight
OD600 (absorbance) protocol: Wavelength: 600nmMeasurement interval time: 8minRun time: 18hGFP (fluorescence) protocol: Excitation: 485 +/- 10Emission: 528 +/- 10Gain: ExtendedRFP (fluorescence) protocol: Excitation: 535 +/- 20Emission: 600 +/- 20Gain: ExtendedLuminescence protocol: Gain: 118Integration time: 1sOD600 + GFP protocol script:Measurement interval time: 8minRun time: 18hOD600 + luminescence protocol script:Measurement interval time: 20minRun time: 20h