eamag/protocols-with-steps huggingface dataset:
TITLE: Peripheral PRV injection - Kidney & Liver Protocol
AUTHORS: Clara Huesing, Heike Muenzberg, Andrea Zsombok, Andrei Derbenev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Pseudorabies viral injections allow for retrograde tracing with fluorescent markers. This protocol demonstrates how to inject into the target organ (kidney or liver) in order to visualize and identify the neural inputs that innervate these organs. </div></div>

[STEPS]
TITLE: EMP 16S rRNA PCR
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
The 16S protocol detailed here is designed to amplify prokaryotes (bacteria and archaea) using paired-end 16S community sequencing on the Illumina platform. Primers 515F–806R target the V4 region of the 16S SSU rRNA.

[BEFORE_START]
The primer sequences without linker, pad, barcode, or adapter are as follows:

Updated sequences: 515F (Parada)–806R (Apprill), forward-barcoded:
FWD:GTGYCAGCMGCCGCGGTAA; REV:GGACTACNVGGGTWTCTAAT

Original sequences: 515F (Caporaso)–806R (Caporaso), reverse-barcoded:
FWD:GTGCCAGCMGCCGCGGTAA; REV:GGACTACHVGGGTWTCTAAT

Note: Primers used for PCR reaction(s) are determined by project and or lab.

[STEPS]
SECTION: Preparing PCR Hood
2. Decontaminate a PCR hood with DNA Away and Ethanol.
SECTION: Preparing PCR Hood
3. Place materials inside of the PCR Hood (refer to materials section).
SECTION: Preparing PCR Hood
4. Turn on the UV setting for at least 30 min.

SECTION: Preparing Master Mix
6. Pour PCR water into a 50mL conical and make the master mix (specific to  project and or number of samples).
SECTION: Preparing Master Mix
5. Label unskirted plate(s) with the following: Project, Extraction Name, Initials, Date, & Plate Number
SECTION: Preparing Master Mix
7. Using a 200uL 12-channel pipette, pipette 22 µL of master mix into each well on each unskirted plate.

 


SECTION: Adding Reverse Primer
8. Once finished, retrieve EMP 10 micromolar (µM) Reverse Primer Plate and DNA plate. Let thaw and spin down.

SECTION: Adding Reverse Primer
9. Add 1 µL of Reverse Primer according to the plate map specific for the project and sample(s).

SECTION: Adding Sample(s)
10. Add 2 µL of sample according to the plate map specific for the project and sample(s).

SECTION: Adding Sample(s)
11. Seal everything with PCR and or freezer seals and clean the PCR hood.
SECTION: Adding Sample(s)
11.1. Return sample(s), reagents, and EMP Reverse Primer plate to the appropriate freezer.
SECTION: Adding Sample(s)
12. Spin down PCR plate(s).
SECTION: Running Thermocycler
13. Place plate(s) on the thermalcycler and start an amplification protocol.
SECTION: Running Thermocycler
14. Once the PCR has finished on the thermalcycler, place PCR plate(s) in a -20 °C freezer or run a gel.

SECTION: Running Thermocycler
13.1. Use the following thermocycler conditions as a part of this earth microbiome project (EMP) protocol:
 

1. Prepare PCR cocktail calculations and a plate map for all PCR reactions using barcoded primers.

The following calculations must be made based on the number of samples to be amplified:

  
Note: The numbers used above are only for demonstration purposes. If calculating the amount of PCR reagents necessary for a set of PCR reactions, calculate using the demonstrated concentrations and volumes per reaction. Typically, every 6 triplicates (or 6 samples + NTCs) requires an input of 25 samples. Therefore, 1 unskirted plate of PCR reactions equates to 100 samples (as the required input to the PCR cocktail calculator as shown above).
TITLE: Striatal Mitochondria Isolation Protocol
AUTHORS: Livia Hecke Morais

[DESCRIPTION]
Striatal Mitochondria Isolation Protocol used in the Mazmanian lab for Seahorse experiments and others

[STEPS]
SECTION: Reagents
1. Reagents

 
500 mL of MSHE (pH 7.2) for isolation

●     210 mM mannitol  (Sigma, Cat #M1902) (19.13 g) 
 
●     70 mM sucrose (Sigma, Cat #S0389) (11.98 g)

●      5 mM HEPES (Sigma, Cat #H3537) (2.5 mL of 1M stock)
 
●     1 mM EGTA (Sigma, Cat #E3889) (2 mL of 250 mM stock)

●      0.2% fatty  acid-free (Fraction V) Bovine-serum albumin (BSA) (1 g, or 10 mL of 10% stock) (Sigma, Cat #A3803)

Measure all the powders into a 500 mL bottle.
Fill the bottle to about 400 mL with tissue culture grade water.
pH to 7.2 at 37˚C with 1M KOH solution while stirring.
Pour contents of 500 mL bottle into a 500 mL graduated cylinder. Fill up to 500 mL with tissue culture grade water.
Filter through 0.22 µm vacuum filter into a 500 mL receiver.
Store at 4˚C.
Prepare it fresh the day before experiment. Or freeze it for future use (maximum of 3 months). Once you defrost it, adjust the pH. 
Also prepare, 250 mL of MSHE without BSA

10% Digitonin (in TC-grade water). (Sigma, Cat #D5628)
Bring to 95˚C in the heat block to dissolve digitonin.
Store at 4˚C.
SECTION: Isolation protocol
2. Pre-chill a 15 mL glass Dounce homogenizers and pestles to 4˚C on ice. Wash the glass tubes with MSHE.
After dissection transfer tissue to the 15 mL dounce homogenizer containing MSHE.
Homogenize with 10 strokes of the pestle.
Transfer the tissue homogenate back into the 2 mL Eppendorf tube.
Centrifuge at 2000g for 3 minutes at 4˚C.
Aliquot the filtered supernatant into chilled 2 mL Eppendorf tubes.
Centrifuge the 2 mL Eppendorf tubes at 12,000g for 10 min at 4˚C.
While waiting for the spin to complete, place 10% digitonin in the heat block to bring it back into solution. Once dissolved, remove it from the heat block to cool before using.
Add 40 µL of 10% digitonin into 20 mL of MSHE (for 0.02% w/v). Scale down if you have less than 10 tubes.
Aspirate supernatant (leave the white layer). Resuspend pellets in 700 µL of the digitonin + MSHE solution (400 µL first then 300 µL). 
Centrifuge again at 12,000g for 10 min at 4˚C.
Aspirate most of the white layer (the light fluffly layer that sits atop the dark brown mitochondria) of the pellet. Resuspend in 700 µL of MSHE.
Centrifuge again at 12,000g for 10 min at 4˚C.
Aspirate the supernatant and remaining white layer of the pellet.
Resuspend the pellet in 30 uL of MSHE (change final volume or buffer as needed).
TITLE: Serotyping 
AUTHORS: lydiariver  

[DESCRIPTION]
Serotyping of Streptococcus agalactiae isolates was carried out by ImmuLex® Strep-B Latex (Statens Serum Institut, Copenhagen, Denmark), classifying them in serotypes 1a,1b, II, III, IV, V, VII, VIII, or IX.  

[STEPS]
1. Use one colony from a 5-10% blood agar plate.
2. Add one drop (10l) of latex reagent on a glass slide.
 

3. Apply 10l phosphate buffer saline next to the drop of latex and suspend one
colony from the agar plate into the drop of saline. 
 



4. Mix the two drops
 

7. 
  

5. Spread to cover the area of the circle.
6. Observe for agglutination within 5-10 seconds.
TITLE: Cell cryopreservation
AUTHORS: PMAT0001 

[STEPS]
?. Gently detach cells from the tissue culture flask in the following manner:
?. Aspirate media
?. Wash with   PBS, then aspirate.
2 mL
?. Wash with   trypsin.
2 mL
?. Incubate for  at  .
37 °C
?. Resuspend the cells in  of DMEM with FBS. Aliquot into a centrifuge tube. Of course, if there is a T25 flask, resuspend in lesser volume.
10 mL
?. Aliquot a small amount of the mixture (about  ) for cell counting. Ideally, cell viability should be in excess of 90% in order to achieve a good recovery after freezing. Keep at - We need 1 x 106 - 5 x 106 cells for freezing.
200 µl
-4 °C
?. Centrifuge the cell suspensions at 1500rpm for
?. Discard supernatant carefully
?. Add about   of freezing media into the tube and re-suspend.
3 mL
?. Freezing media preparation:- 10% DMSO, 20% FBS, 70% appropriate cell culture medium (with serum + antibiotics)(e.g 100uL of DMSO + 200uL FBS + 700uL cell culture medium with FBS and antibiotics)
?. Dispense the aliquots of cell suspension into the cryogenic storage vials.
?. Cool the vials gradually as follows:  for   overnightLiquid Nitrogen
-20 °C
-80 °C
TITLE: Purification of mCh-WIPI2d-IDR (364-426aa)
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of mCherry WIPI2d-IDR.

[STEPS]
SECTION: Purification procedure
1. To purify mCherry-WIPI2d IDR (364-426aa) (available from Addgene), fuse the corresponding coding sequence of WIPI2d  to a N-terminal 6xHis-TEV-mCherry-tag through cloning into a pET-DUET1 vector (available from Addgene). 

SECTION: Purification procedure
2. After the transformation of the pET-DUET1 vector encoding 6xHis-TEV-mCherry-WIPI2d-IDR in E. coli Rosetta pLysS cells(Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C. 

SECTION: Purification procedure
3. Once the cells reach an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 

SECTION: Purification procedure
15. Store the proteins at -80 °C.

SECTION: Purification procedure
14. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
11. Elute the proteins with SEC buffer. 

SEC buffer:
 
 
SECTION: Purification procedure
10. Pool fractions containing the 6xHis-TEV-mCherry-WIPI2d-IDR, concentrate using a 10 kDa cut-off Amicon filter (Merck Millipore) and load onto a pre-equilibrated S75 Increase 10/300 column (Cytiva). 

SECTION: Purification procedure
9. Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM). 

SECTION: Purification procedure
8. After bind His tagged proteins to the column, was the column with three column volumes of wash buffer. 

Wash buffer: 
 
 
SECTION: Purification procedure
7. Filter the supernatant through an 0.45 µm filter and load onto a pre-equilibrated 5 ml His-Trap HP column (Cytiva). 

SECTION: Purification procedure
6. Clear the lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
5. Sonicate the cell lysates twice for 30 s. 

SECTION: Purification procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer, complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)). 

Lysis buffer:
 
 
SECTION: Purification procedure
13. Pool the fractions containing purified mCherry-WIPI2d-IDR. 

SECTION: Purification procedure
12. Analyse the fractions by SDS-PAGE and Coomassie staining.
TITLE: Detection of anti- SpA antibodies in egg white tested by  double immunodiffusion (Ouchterlony) technique.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Keyhole limpet hemocyanin (KLH) is a cooper-containing protein comprising of subunits with MW of 400 kDa. This protein is found in the hemolymph of the sea mollusk Megathura crenulata. It has the ability to enhance the host’s immune response by interacting with monocytes, T cells and macrophages. KLH has been used primarily as a carrier for vaccines and antigens [1]. It was found that chicken immunized with KLH bound peptide raised an anti-KLH immunoresponse [2]. This can be tested by a single method such as the Ouchterlony technique.</div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Aarntzen EH, de Vries IJ, Göertz JH, et al. Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters.</span><span style = "font-style:italic;">Cancer Immunol Immunother</span><span>. 2012;61(11):2003-2011. doi:10.1007/s00262-012-1263-z</span></div><div class = "text-block">2. Justiz Vaillant AA, Anderson MF, Smikle M, Wisdom B, Mohammed W, et al. (2013) Development of Anti HIV Gp120 and HIV Gp41 Peptide Vaccines. J Vaccines Vaccin 4: 206. doi: 10.4172/2157-7560.1000206 </div></div>

[STEPS]
?. Detection of anti-keyhole limpet hemocynin antibodies by double immunodiffusion is carried out.
?. Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
?. Initially, aliquots of 25 µl each of KLH in concentration of 1 mg/ml are applied to the centre well.
?. The peripheral wells are filled with 25 µl of isolated chicken IgY (1 mg/ml) in PBS pH 7.4.
?. The gels are incubated at RT for 48−72 hours.
?. After that the gels are examined for precipitin lines.
?. An anti-KLH developed in cat is included as positive control and turtle serum as a negative control.
?. The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
TITLE: MicroCT protocols for scanning egg capsules of Hexaplex trunculus
AUTHORS: Eva Chatzinikolaou, Kleoniki Keklikoglou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Micro-computed tomography (micro-CT) is a high-resolution 3D-imaging  technique which is now increasingly applied in biological studies  focusing on taxonomy and functional morphology. The creation of virtual  representations of specimens can increase availability of otherwise  underexploited and inaccessible samples. This protocol aims to standardise micro-CT scanning procedures for the egg capsules of the marine gastropod species </span><span style = "font-style:italic;">Hexaplex trunculus. </span></div></div>

[STEPS]
?. [Sample preparation]
Fixation of egg capsules of Hexaplex trunculus in 5% formaldehyde buffered with seawater.
?. [Sample preparation]
Wash egg capsules with distilled water and dehydrated them with ethanol in gradually increasing concentrations (20%, 50%, 70%, 96%).
?. [Sample preparation]
Placement of samples inside a plastic Falkon tube with 96% ethanol as a scanning medium.
?. [microCT scanning]
?. [microCT scanning]
Scanning parameters for embryos and juvenile Hexaplex trunculusVoltage: 80 kVCurrent :124 μAFilter: aluminium Pixel size: 13.79 μmCamera binning: 2 × 2Exposure time: 1435 ms Rotation: 360° Rotation step: 0.40º Frame averaging: 3
?. [Images reconstruction]
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium).
?. [Images reconstruction]
Upload projection images.
?. [Images reconstruction]
Perform X-Y alignment.
?. [Images reconstruction]
Reconstruction parametersSmoothing: 2Ring artifact correction: 20Beam hardening correction: 59%Attenuation coefficients: 0 - 0.064Save as: 16-bit TIFF images
?. [Sample preparation]
Staining of egg capsules with 1% iodine in 96% ethanol.
?. [Sample preparation]
Washing of stained samples with 96% ethanol.
TITLE:      ELISA for quantification of IL-22 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>Different cells in both innate and acquired immunities produce IL-22, but the primary sources are T cells. Th22 cell is a new line of CD4+ T cells, which differentiated from naive T cells in the presence of various pro-inflammatory cytokines including IL-6. IL-22 inhibits IL-4 production. It also has essential functions in mucosal surface protection and tissue repairy. </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-22 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-22 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-22 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-22 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-22 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-22 standard dilutions and the human IL-22 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Separation of reovirus particles by gel electrophoresis</p>

[BEFORE_START]
<p><strong>Materials and Reagents</strong></p>
<p> </p>
<p>-1M Tris Acetate pH 7.2</p>
<ul>
<li>MW = 121.1g/mol</li>
<li>Add 60.5 g Tris base to 300 ml ddH<sub>2</sub>O</li>
<li>pH to 7.2 with glacial acetic acid</li>
<li>Fill to 500 ml total volume<sub><br /></sub></li>
</ul>
<p> </p>
<p>-10X TAE pH 7.2</p>
<ul>
<li>125 ml 1M Tris acetate pH 7.2</li>
<li>50 ml EDTA pH 8.0</li>
<li>75 ml ddH<sub>2</sub>O</li>
<li>Dilute to 1x as needed and filter 0.45 uM to remove any potential debris</li>
</ul>
<p>-UltraPure<sup>TM</sup> Agarose, Thermo Scientific</p>
<p> </p>
<p>-NOVEX® Colloidal Blue Staining Kit (follow directions for [high] Tris Glycine)</p>
<p> </p>
<p>-Reovirus 2x Loading Dye</p>
<ul>
<li>5% Ficoll 400 (0.5g)</li>
<li>05% Bromophenol blue (50 mg)</li>
<li>2 ml 10X TAE pH 7.2</li>
<li>Fill to 10 ml with ddH<sub>2</sub>O</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
An alternative protocol for isolated astrocyte cell culture preparation. Dissociation, preparation and plating of mice cortex neurons and glia cells on flasks. Isolatioin process of the culture from neurons, to obtain an isolated astrocyte culture. Plating isolated astrocytes on MEA.

[STEPS]
SECTION: Sample collection
?. 
SECTION: Cell lysis
?. 
SECTION: Cell preparation
?. 
SECTION: Cell culture
?.
TITLE: Polymer-brush-bilayers-equilibrium
AUTHORS: Mike Edwards

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">By means of statistical mechanics of polymers and density functional  theory (DFT), polymer brush bilayers are investigated at thermal  equilibrium. Density profiles show how brushes balance compression and  interpenetration when they come into contact. Normal pressure indicates  that substrates might repel, attract or even do not sense each other  (though the brushes interpenetrate) upon varying system parameters. The  results, based on that it takes deformations of both brushes into  account, suggests that existing theories about polymer brush bilayers  (based on blob picture) need highly likely to be reviewed.</div></div>

[STEPS]
TITLE: Intaspecific aggression assay for Nylanderia fulva
AUTHORS: Edward LeBrun

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for conducting intraspecific aggression assays with the tawny crazy,</div><div class = "text-block"><span style = "font-style:italic;">Nylanderia fulva</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
TITLE:   Direct ELISA for investigating the binding of Peptostreptococcal Protein-L  to immunoglobulins.
AUTHORS: Angel  Justiz-Vaillant, Monica F. Smikle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Peptostreptococcal protein L is an immunoglobulin-binding protein that interacts with the Fab region of many mammalian immunoglobulins [1].</div><div class = "text-block">References</div><div class = "text-block">1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175 </div></div>

[STEPS]
?. This ELISA is used to study the interaction of Peptostreptococcal protein-L (SpL) with diverse immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins or 50 µl of any animal sera in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-protein-L conjugate diluted 1:3000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 2 SD.