eamag/protocols-with-steps huggingface dataset:
TITLE: Mitophagy induction using Difereprone
AUTHORS: Louise Uoselis

[DESCRIPTION]
Mitophagy induction in HeLa cells using Difereprone (DFP)

[STEPS]
SECTION: Day 1
1. Seed cells, aiming for a confluency of 80-90% at the time of treatment the next day. 
SECTION: Day 2
2. Feed cells for 60 min in an appropriate volume of standard growth media. 

SECTION: Day 2
3. During the feed, warm up the difereprone (DFP) stock aliquot in a 37 deg C waterbath. Centrifuge the stock tube after thawing to ensure the DFP has returned to solution. 
SECTION: Day 2
4. To start the treatment, replace the media in each well with standard growth media that contains 1 mM DFP. 
SECTION: Day 2
5. Harvest the samples after the desired treatment times.
TITLE: BHI/LB + v2 salts media
AUTHORS: Matthew Haines

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Vibrio natriegens  </span><span>grows well media containing v2 salts e.g. BHI + v2 and LBv2 (</span><a href="http://2018.igem.org/Team:Marburg/Results" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">link</span></a><span>). Furthermore, such media is routinely used for culturing this organism (Weinstock et al., 2016). However, care must be taken during preperation not to autoclave v2 salts and media together. This protocol first generates separate solutions before sterilising and combining them.</span></div></div>

[STEPS]
?. [Prepare stock salt solutions]
Prepare the following salt solutions at the given concentrations:
[NaCl]
[KCl]
[MgCl2.6H2O]
?. [Prepare media]
Dissolve   or  in   ddH2O in a 1 L graduated bottle.
[BHI dry medium]
[LB Broth (Miller)]
400 mL
?. [Sterilise and combine]
Sterilise all solutions by autoclaving.
?. [Sterilise and combine]
Under sterile conditions, transfer the following volumes of stock salt solutions to the BHI media:  ABCD1SaltStock solution (M)Volume (mL)Final concentration (mM)2NaCl520.42043MgCl2.6H2O111.623.24KCl12.14.2
ABCD1SaltStock solution (M)Volume (mL)Final concentration (mM)2NaCl520.42043MgCl2.6H2O111.623.24KCl12.14.2
?. [Sterilise and combine]
Adjust the volume to   using sterile ddH2O.
500 mL
TITLE: PNGase F Protocol (Non-Denaturing Reaction Conditions)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides. 

This is a generic PNGase F protocol for non-denaturing reaction conditions. It is appropriate for both P0704 and P0708.

   


[BEFORE_START]
Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:

[GUIDELINES]
If using P0704/P0708, we recommend limiting PNGase F to 1/10 (or less) of the total reaction volume to keep the final glycerol concentration equal to (or less than) 5%.
For unit conversion between different suppliers, please reference the Glycobiology Unit Conversion Chart page.

Biology Unit Conversion Chart
Reagent companies differ in how a unit of enzyme is defined. This chart can be used to help determine how a unit of enzyme from one company compares to a unit of enzyme from NEB. All enzymes were assayed using NEB's assay protocols as a means of normalization (NEB Assay).
EnzymeCompanySelling Conc. (U/ml)Units /Vialµl/VialNEB Assay (U/ml)NEB Assay Units /Vialµl Conversion (1 NEB µl = x Company µls)PNGase FNEB (NEB #P0704/P0705)500,00015,00030500,00015,0001Prozyme (GKE-5006A)2.50.140150,0006,0003.3Prozyme (GKE-5020B, Ultra)100.440500,00020,0001QA Bio (E-PNG01)50.360200,00012,0002.5Sigma (P7367)500505090,0004,5005.5
 

[STEPS]
SECTION: Non-Denaturing Reaction Conditions:
1. 
 
Combine 1 µg - 20 µg, 2 µL and H2O (if necessary) to make a 20 µL total reaction volume.
SECTION: Non-Denaturing Reaction Conditions:
2. Add 2 µL - 5 µL, mix gently.
SECTION: Non-Denaturing Reaction Conditions:
3. Incubate reaction at 37 °C for 240 min - 1440 min.
 

SECTION: Non-Denaturing Reaction Conditions:
4. Analyze by method of choice.
 

None. Combine 1-20 µg of glycoprotein, 2 µL and H2O (if necessary) to make a 20 µl total reaction volume.

None. Add 2 µL to 5 µL, mix gently.

None. Incubate reaction at 37 °C for 240 min - 1440 min.

 

None. Analyze by method of choice.
 

None. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:
TITLE: Protocol details for autopsy specimens (WUSTL)
AUTHORS: Reyka Jayasinghe

[DESCRIPTION]
protocol details for autopsy specimens

[STEPS]
SECTION: Sample Collection Strategy
1. The tissue is transported on ice to the lab for processing and storage.  

Larger samples are sectioned into small segments and placed into 2 mL cryovial before dropping into liquid nitrogen.  

Vials are immediately placed in -80° freezer
TITLE: Immunofluorescence and Cell Counting
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains Immunofluorescence and Cell Counting for lines ND1014, N1, and ND27760 from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [Immunofluorescence]
Fix cells in 4% paraformaldehyde in PBS .
?. [Immunofluorescence]
Block cells in 5% normal goat serum and 0.2% Triton X-100.
?. [Immunofluorescence]
Dilute primary antibodies in 5% normal goat serum. Primary antibodies are listed in the "Guidelines".
?. [Immunofluorescence]
Incubate samples with primary antibodies  at .
4 °C
?. [Immunofluorescence]
Counterstain with DAPI.
?. [Immunofluorescence]
Mount on glass slides using ProLong antifade.
?. [Cell Counting]
Randomly select 10 different fields to use for counting the number of DAPI-positive cells expressing specific markers in ImageJ. Perform this with at least three independent experiments.
?. [Immunofluorescence]
Stain using Cy3‐ and Alexa Fluor 488‐labeled secondary antibodies.
TITLE: Fixation of fluorescent E.coli by Mannik 
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Per Jaana Mannik Communication, June 1st 2020:</div><div class = "text-block">For the fixation of FtsZ-mNG cels, we have been using a mix of paraformaldehyde (PFA) and glutaraldehyde (see mu protocol below). In the case of FtsZ, we will lose a specific Z-ring-like structure using only PFA; the addition of glutaraldehyde helps to preserve it.</div><div class = "text-block">If your protein of interest is not sensitive to stress response, the response fixation with only PFA should work well. fixation with PFA should maintain fluorescence signal as it is. </div></div>

[STEPS]
?. [Fixation of FtsZ-mNG E.coli cells]
Fix cells directly in growth medium by addition of paraformaldehyde (PFA) to final concentration 2.8% (vol/vol) and glutaraldehyde (GH) to 0.04% (vol/vol) for 15 minutes at RT (work with PFA and GH under the chemical hood!)
?. [Fixation of FtsZ-mNG E.coli cells]
Transfer the cells to epis and spindown at 6000 rpm for 1:30 minutes
?. [Fixation of FtsZ-mNG E.coli cells]
Remove supernatant (under the chemical hood; collect supernatant to PFA/GH waste bottle)
?. [Fixation of FtsZ-mNG E.coli cells]
Wash 2x with PBS: add 1ml of 1xPBS and resuspend the pellet, spin down at 6000 rpm for 1:30 minutes, remove super. Repeat the step. (RT or 4oC). The cells can be stored up to a month at 4oC. Keep the cells in the dark
TITLE: 51Cr Release Cytotoxicity Assay for murine CAR T cells
AUTHORS: Tamer B Shabaneh, Andrew R Stevens

[DESCRIPTION]
A standard cell-based assay to assess the function of murine CAR T cells, which we regularly perform at the end of the process of generating CAR T cells (see "Retroviral transduction of primary murine CD8 T cells").

[STEPS]
SECTION: Labeling target cells with 51Cr (day 0)
1. For each adherent tumor cell line, plate 5x105 cells / 3mL (previously growing in log phase) in one 6-well-plate well; Incubate cells for ~3hr to rest.
SECTION: Labeling target cells with 51Cr (day 0)
2. In the Hot Lab, label the target cells with 50 μL 51Cr (vial <1wk old), 100μL (1-2wk old), or 150μL (>2wk old). Place in Hot Lab incubator overnight.
 

SECTION: Processing effector cells (day 1)
3. From each transduced T cell  culture, remove 9x105 cells, wash with fresh mouse T cell media (mTCM) for 6min at 400rcf, and resuspend in 3mL mTCM.
 

SECTION: Processing effector cells (day 1)
5. Begin processing the target cells. To remove extracellular 51Cr, briefly wash the adherent lines with 5mL Cell Dissociation Buffer (0.5mM EDTA in PBS). Repeat the wash. Dispose the 51Cr-containing media appropriately.
 

SECTION: Processing effector cells (day 1)
4. In triplicates, transfer 150μL (4.5x104 effector cells) to U-bottom 96-well-plate wells (in columns 1, 2, 3). Pipet mTCM (100uL/wel) into the remainder of the row (4-12); prepare 3 serial dilutions (1:3) by pipetting 50uL to the respective 100uL volumes.
 

SECTION: Processing effector cells (day 1)
4.1. Example layout: 
 
  

SECTION: Processing effector cells (day 1)
6. Count the cells using a hemocytometer. Adjust volume to 1E4 cells / mL mTCM.
Transfer 100μL (1E3 target cells) to each well on the respective half of the plate already containing 100uL of effector CAR-T cells. 
SECTION: Processing effector cells (day 1)
5.1. To dissociate target cells, add 3mL Cell Dissociation Buffer to each well, incubate at 37C for 10-15 mins. Add 3mL mTCM; centrifuge at 400rcf for 4min.
SECTION: Processing effector cells (day 1)
7. Finally, transfer 100μL target cells to an extra row. Add 100μL of soap (NP40+Trypan blue) to generate a positive control. Add 100μL mTCM to generate a negative control.
SECTION: Processing effector cells (day 1)
8. Centrifuge plate at 800rpm for 3mins; incubate at 37C for 24 hours.
SECTION: Reading scintillation plate
9. Harvest 30uL supernatant from each plate; carefully transfer onto respective Luma plate wells.
Dry the plate completely for ~24 hours. 
SECTION: Reading scintillation plate
10. Acquire data on the plate scintillation counter per respective protocol.
TITLE: Mycology media
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
Mycology media

[STEPS]
1. WA+C agar 
Water agar + chloramphenicol (氯黴素 kill bacteria): As an environmental isolating media

 

2. PDA+C agar
potato dextrose agar + chloramphenicol (氯黴素 kill bacteria): Use when WA+C agar is not working

 

3. Emerson YpSs Agar: For chytrids

 

4. 10% Unclarified V8 Agar
Combine 100 ml V8 juice and 900 ml of distilled water.
Add 1g CaCO3and 0.05 g b-sitosterol and mix well. Leave stir bar in the flask for later mixing.
Add 15g of agar.
Autoclave at 15 psi for 20 minutes.
Stir medium while dispensing to insure good mixing of CaCO3.
20% : 200 ml V8, 800ml d. water, 2g CaCO3, and 0.05g b-sitosterol.
15% : 150 ml V8, 850 ml d. water, 1.5g CaCO3, and 0.05g b-sitosterol.
Reference: Miller, P. M. 1955. V-8 juice agar as a general purpose medium for fungi and bacteria. Phytopathology 45:461-462.
5. SceSel+ medium (Kaltseis et al.,2009)
For isolation of Scedosporium
TITLE: PNA Oligomer Synthesis
AUTHORS: Cathy  Miller

[DESCRIPTION]
PNA is a pseudo-peptide backbone instead of the ribonucleic acid-phosphate backbone, retaining the original 4 bases. Nielsen et al in Rose Laboratory of the University of Copenhagen used a simple computer DNA model to successfully design and synthesize PNA and PNA in 1991. The synthesis of PNA is first to synthesize the monomers, and then oligomerize the PNA monomers according to the standard peptide synthesis method. The structural unit of PNA is modified amino acid, so it can be prepared in large quantities by peptide synthesis, and it can also be prepared by chemical synthesis by attaching functional groups to the N-terminus or C-terminus. PNA oligomers are prepared from protected PNA monomers by solid-phase synthesis, and their protection strategies, condensation conditions, deprotection methods and purification methods are similar to those of peptide synthesis.

Creative Peptidesteam has accumulated rich experience in PNA chemistry, we can provide two chemical synthesis methods to synthesize PNA oligomers, including Boc chemistry and Fmoc chemistry. The entire synthesis process will be implemented in accordance with strict quality control protocols, and we are committed to synthesizing high-quality, high-precision, and high-activity PNA products to meet customer expectations.

[STEPS]
TITLE:        ELISA for quantification of IL-4 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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.  </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">The immunoregulatory cytokine IL-41 (also known as meteorin-like protein) is expressed at high levels in the synovium of patients with psoriatic arthritis (PsA).[2]</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 class = "text-block"><span>2.Onuora S. Novel cytokine, IL-41, linked with PsA.</span><span style = "font-style:italic;">Nat Rev Rheumatol</span><span>. 2019;15(11):636. doi:10.1038/s41584-019-0314-7 </span></div></div>

[STEPS]
?. An anti-human IL-4 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-4 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-4 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-4 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-4 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-4 standard dilutions and the human IL-4 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Ligation Protocol with T4 DNA Ligase
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting the sugar backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.</div><div class = "text-block">The majority of ligation reactions involve DNA fragments that have been generated by restriction enzyme ligation (See Protocols). Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are compatible, meaning that the overhanging base pairs on the vector and insert are complementary, the two pieces of DNA connect and ultimately are fused by the ligation reaction.</div></div>

[STEPS]
?. Thaw the T4 DNA Ligase Buffer and resuspended at room temperature.  'Pro-tip': Alicuote the 10x buffer less concentrated so when thawing the DTT gets soluble more easily.
?. Set up the reaction in a microcentrifuge tube on ice (T4 DNA Ligase should be added last.):  AB1COMPONENT20 μl REACTION2T4 DNA Ligase Buffer (10X)*2 μl3Vector DNA (4 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free waterto 20 μl6T4 DNA Ligase 1 μl
AB1COMPONENT20 μl REACTION2T4 DNA Ligase Buffer (10X)*2 μl3Vector DNA (4 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free waterto 20 μl6T4 DNA Ligase 1 μl
?. Gently mix the reaction by pipetting up and down and microfuge briefly.
?. For cohesive (sticky) ends, incubate at 16ºC overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16ºC overnight or room temperature for 2 hours.
?. Heat inactivate at 65ºC for 10 minutes.
?. Chill on ice and transform 1-5 μL of the reaction into 50 μL competent cells (See Protocols for Transformation).
TITLE: E. coli Plating Quantification
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to serially dilute and plate cultured </span><span style = "font-style:italic;">E. coli </span><span>K-12 in suspension for quantification.</span></div></div>

[STEPS]
?. [Prepare Serial Dilutions]
Transfer of stock solution to 2 mL centrifuge tube.
100 µl
?. [Prepare Serial Dilutions]
Add DI water to the same centrifuge tube to make a dilution that is one-tenth the concentration of the stock solution.
900 µl
?. [Prepare Serial Dilutions]
Using of the dilution you just made, make another dilution that is one-tenth the concentration of the second solution.
100 µl
?. [Prepare Serial Dilutions]
Repeat 6 times until you have a solution that is diluted by 106.
?. [Plate]
Choose a dilution that you feel is appropriate and dispense of that dilution on agar plate.
100 µl
?. [Plate]
Evenly spread the cell suspension over the agarose gel with inoculation loop.
This may be different from how you have streaked before. We are wanting the solution evenly spread so that we can count how many colonies are formed after incubation.
?. [Plate]
Wrap the agar plate's edge in parafilm.
If you have not worked with Parafilm, ask a TA for help in wrapping your plate.
?. [Plate]
Wait 2 minutes, then flip the plate upside-down for incubation.
?. [Plate]
Label the plate with:Group numberDateDilution used (10^x)
?. [Incubate]
Incubate at over night.
Performed by T.A.
?. [Incubate]
Take image of incubated plate.Include this image in the lab report results section.
Performed by T.A.
?. [Quantify Concentration]
Using the image of your plate, count the number of colonies formed. This can be done by hand or using an image processing software such as ImageJ.
?. [Quantify Concentration]
Calculate the original E. Coli concentration using the following equation:Include the result of this calculation in the lab report.
TITLE: The Pair Test - a computerised measure of learning and memory
AUTHORS: Sarah Buck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Pair Test offers improved diagnosis, with more precise indication on the nature of the memory deficit and the underlying processing impairment in patients with known or suspected pathology in the temporal lobe. The advantages of the Pair Test are 1) a balanced paradigm allowing comparison between verbal and non-verbal memory, 2) assessment of learning, delayed recall and recognition using one tool, 3) paired-associate learning tool to assess cognitive processes that are dependent on the hippocampal network, and 4) the use of a tablet-based application allowing controlled administration process and engaging approach to neuropsychological testing. The test provides behavioural evidence of the integrity of the hippocampi and its interaction with the neocortical learning system.</div><div class = "text-block"><span>Buck et al., (2020). The Pair Test: A computerised measure of learning and memory. </span><span style = "font-style:italic;">Behavior Research Methods</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
TITLE: Exp_01
AUTHORS: José Eduardo De la Cruz Luna

[DESCRIPTION]
Creacion del primer experimento

[STEPS]
SECTION: Exp_01
1. Conectar los arduinos, resistencas y sensores(DS18B20) al protoboard
SECTION: Exp_01
2. Prepara el codigo que pueda capturar los datos del agua con los sensores
SECTION: Exp_01
3. Hervir el agua a 100ºC
SECTION: Exp_01
4. Colocar un sensor en el asa y el otro dentro de la taza
SECTION: Exp_01
5. Vertit el agua hirviendo a la taza
SECTION: Exp_01
6. Comprobar que la laptop esta capturando los datos
SECTION: Exp_01
7. Observar el experimento hasta que ambos sensores lleguen a temperatura ambiente
TITLE: Fungal Extraction from Beetles
AUTHORS: You Li, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to extract fungal symbionts from beetles. </div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]