eamag/protocols-with-steps huggingface dataset:
TITLE: DNA Clean & Concentrator 
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Plasmid DNA</li><li style = "counter-reset:ol0;list-style-type:disc;">Binding Buffer </li><li style = "counter-reset:ol0;list-style-type:disc;">DNA Wash Buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">DNA Elution Buffer or </li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Preparation</span></div><div class = "text-block">Add 24 ml 100% ethanol ( 26 ml 95% ethanol ) to the 6 ml DNA Wash Buffer concentrate. Add 96 ml 100% ethanol ( 104 ml 95% ethanol ) to the 24 ml DNA Wash Buffer concentrate.</div><div class = "text-block">Perform all centrifugation at ≥ 10000 x g.</div><div class = "text-block"><span style = "font-weight:bold;">Implementation</span></div><div class = "text-block">In a 1,5 ml microcentrifuge tube, add 2 - 7 volumes of DNA Bindung Buffer to each volume of DNA sample. Mix briefly by vortexing.</div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Application</td><td rowspan = "" colspan = "" style ="display : table-cell;">Bindung Buffer : sample</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">plasmid, genomic DNA ( &gt; 2 kb)</td><td rowspan = "" colspan = "" style ="display : table-cell;">2 : 1</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">PCR product, DNA fragment</td><td rowspan = "" colspan = "" style ="display : table-cell;">5 : 1</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">ssDNA</td><td rowspan = "" colspan = "" style ="display : table-cell;">7 : 1</td></tr></table><div class = "text-block">Transfer mixture to a Zymo- Spin Column in a Collection Tube.</div><div class = "text-block">Centrifuge for 30 seconds. Discard the flow-through.</div><div class = "text-block">Add 200 µl DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat the wash step.</div><div class = "text-block">Add ≥ 6µl DNA Elution Buffer or water directly to the column matrix and incubate at room temperature for one minute. Transfer the column to a 1,5 ml microcentrifuge tube and centrifuge for 30 seconds to elute the DNA. </div></div>

[STEPS]
TITLE: Competitive ELISA to study the inhibition of the SpA- binding to Ab-3 by Ab-2.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This immunoassay tested the ability of anti-anti-SpA (Ab-2) to inhibit or interfere with the binding of anti-anti-anti-SpA (Ab-3) to the original antigen- staphylococcal protein A (SpA). It is a confirmation test of the functional capacity of Ab-2. In this ELISA, both Ab-2 and the antigen SpA compete for binding to Ab-3. When Ab-2 is present, it blocks the binding of HRP-labeled SpA to Ab-3, resulting in inhibition of reaction color development. </div></div>

[STEPS]
?. Prepare materials and ELISA buffer solutions and reagents.
?. Pipette 53 μl of purified Ab-3 (100 μg/ml) mixed with 2.8 ml of coating buffer into each well.
?. Incubate the microplate at 37°C for 4 hrs.
?. Aspirate the contents of the wells.
?. Fill each well with an  appropriately diluted washing solution and aspirate.
?. Wash the microplate 3 times.
?. Pipette 53 μl of serial dilutions of pooled Ab-2 (100 μg/ml) in triplicates.
?. Incubate the  microplate at RT for 1 hr.
?. Rewash the microplate filling each well with 100 μl of washing buffer.
?. Pipette 53 μl of  commercially available peroxidase-labeled SpA conjugate (Sigma-Aldrich) diluted 1:5000 to each well.
?. Reincubate the microplate at RT for 1 hr.
?. Repeat step 5.
?. Pipette 53 μl of TMB  (Sigma-Aldrich) to each well.
?. Incubate in the dark at RT for 14 min.
?. Measure absorbance at 450 nm in a microplate reader and analyze the results
TITLE: Proteomics :On-bead/in-solution digestion and phosphopeptide enrichment
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Proteomics :On-bead/in-solution digestion and phosphopeptide enrichment

[STEPS]
1. Protein digestion of GST samples was performed following published protocols 
 






2. 
Digestion buffer (50 mM NH4HCO3) was added to
the sample, followed by treatment with 1 mM dithiothreitol (DTT, Thermo
Scientific Cat#R0861) at room temperature for 30 min. Subsequently, 5 mM
iodoacetamide (IAA, Sigma Cat#I1149) was added to the mixture and incubated at
room temperature for 30 min in the dark. 
3. Proteins were digested with 2 µg of lysyl endopeptidase (Wako, Cat# 125-05061) at room temperature (overnight).

4. Further digestion was performed with 2 µg trypsin (Promega, Cat#VA9000) at room temperature
(overnight). In contrast, for phosphopeptide enrichment, proteins were digested
overnight at room temperature with 1 µg of Asp-N (Promega, Cat# VA1160) for samples in solution or 5 µg of Asp-N for the samples
on-beads. 
5. The clean peptides were concentrated using a high-select Fe-NTA
phosphopeptide enrichment kit (Thermo Scientific Cat#A32992) All resulting
peptides were desalted and dried using an HLB column (Waters, Cat#186002034)
and vacuum.
TITLE: Neurospora culture and basic imaging
AUTHORS: gamclaug  

[DESCRIPTION]
Details about culturing Neurospora, imaging, making freezer stocks, and media recipe

[GUIDELINES]
Neurospora spores can travel through the air, so work with the spores/cells in a fume hood to avoid contamination. Only open slants and plates in the fume hood. 

http://www.fgsc.net/Neurospora/neurospora.htmlis a good neurospora resource 

[STEPS]
SECTION: Intro
1. Neurospora spores can travel through the air, so work with the spores/cells in a fume hood to avoid contamination. Only open slants and plates in the fume hood. 

http://www.fgsc.net/Neurospora/neurospora.htmlis a good neurospora resource 
SECTION: Cell Culture
2. Neurospora spores are generally mailed between labs on small pieces of filter paper. To start a new slant tube from them, push the filter paper into the agar at the bottom of the slant tube. Put on the lid, and leave the tube at room temp. There will be plenty of mycelia in 1-2 days. 

To start a new plate, simply insert a sterile applicator stick into the slant tube and then touch that to the center of a plate. The plate can then be left to grow at room temp. This slant tube can be used many times for this for about 2 weeks.

  
After 2 weeks, transfer the cells from the original slant tube to a new slant tube (with the same method as above- except stick the applicator stick into the new slant instead of the plate).
SECTION: Imaging
3. Grow the cells on the plate at room temp until they cover about 2/3 of it. This usually takes about 12h.

Cut out 2cm x 1cm pieces of agar around the edge of the colony (if your intention is to image the apical regions):

  
Put 20uL of Vogel’s Minimal Media in a 15u-Slide 4 well Ibidi chamber (80426), and then put the agar pieces into the wells with cells facing down:

  
For imaging high SNR things like nuclei using agar plates is fine. Agarose plates might be better for dimmer signals.

It can also work to put the agar pieces onto a coverslip, but this is not as stable as the ibidi chambers. It works okay to do this on Ann, but does not work very well on Bronty when taking z-stacks.
SECTION: Making freezer stocks
4. Start with a slant tube that has mycelia growing in it. Pour 2mL of 25% glycerol into the tube, put the cap on, and vortex. Pipette this into cryto tubes and store at -80.

To start a new slant from the freezer stock, use a sterile applicator stick to scrape out a small amount of the freezer stock and put in the middle of the plate. Do not let it thaw, just scrape off the top.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The identity of a nucleotide or the presence of a bulky modification or strand break in an RNA can be determined by several approaches. When the 3′ region of the analyzed RNA is known, extending a (radio)labeled primer by reverse transcription and analyzing the reaction products using denaturing polyacrylamide gel electrophoresis and subsequent (radio)imaging allows mapping of cDNA chain-termination sites. (Based on the design of the experiment, these can be, for example, a strand break, 5′ end, pause-inducing site, or site of dideoxynucleotide incorporation.) This procedure is adapted from the previously published protocol by Walker and Lorsch (DOI: 10.1016/B978-0-12-420037-1.00020-8).

[BEFORE_START]
<div>
<ul>
<li>Prepare 10× annealing buffer: 0.5 M HEPES, pH 7.5 + 1 M KCl.</li>
<li>Prepare 5× extension buffer: 0.5 M Tris, pH 8.0 + 50 mM MgCl2 + 50 mM DTT.</li>
<li>Prepare 1 M NaOH and 1M HCl.</li>
<li>Prepare 2× STOP/loading buffer: 95% deionized formamide, 0.025% SDS, 0.5 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF.</li>
<li>Prepare 5× TBE: 49.5 mM Tris + 49.5 mM boric acid + 2 mM EDTA, pH 8.0.</li>
<li>Use RNase-free water in all solutions.</li>
</ul>
</div>

[GUIDELINES]
<ul>
<li>Estimate the concentration of the target in the sample, whether working with a purified transcript or a complex RNA mixture (e.g. total RNA, poly(A) RNA.). For annealing, RNA substrate and labeled oligo should be equimolar. If primer annealing is problematic because of prominent RNA folding (e.g. if the target is a highly structured RNA), the ratio target:primer can be varied in the range of 1:1 to 1:10 to find the optimal hybridization conditions.</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 19">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The ISOLATE II Biofluids RNA Kit isolates total RNA with minimal amounts of genomic DNA contamination using the supplied Genomic DNA Removal Column. However, additional DNase I treatment may be required in certain cases e.g. the amount of genomic DNA in the sample exceeds the capacity of the Genomic DNA Removal Column, or performing a highly sensitive application.</p>
<p> </p>
<p>The <a href="https://www.protocols.io/view/optional-on-column-dnase-i-treatment-for-isolate-i-f5ibq4e" target="_blank">on-column DNase I digestion results</a> in minimal residual DNA, undetectable in most downstream applications. For the most sensitive applications, DNA digestion in solution is recommended to eliminate even traces of contaminating DNA. Stringent RNase control is needed as well as RNA repurification to remove buffer, salts, DNase I and digested DNA.</p>
</div>
</div>
</div>
</div>

[GUIDELINES]
<div class="page" title="Page 20">
<div class="layoutArea">
<div class="column">
<p>Additional reagents required:</p>
<ul>
<li>
<p>Sodium acetate (3 M, pH 5.2)</p>
</li>
<li>
<p>Ice-cold 70% ethanol</p>
</li>
</ul>
<p> </p>
</div>
</div>
</div>
<p>Please review the Guidelines under <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Genomic DNA removal and total RNA purification from all types of lysate</a> for important details.</p>

[STEPS]
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TITLE: Streptavidin immovilization stressed test
AUTHORS: Jorge Fernández

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol was performed to test the affinity of the protein streptavidin for the nitrocellulose when applied a continuous flow of solvent.</div></div>

[STEPS]
?. Cut the FF80HP nitrocellulose membranes in  2 rectangles of 0.8cm x 4 cm dimensions. Repeat this procedure with the FF170HP nitrocellulose to obtain a total of 4 nitrocellulose pieces.
?. Stamp  the microfluidic design, following the protocol microfluidic channels wax priming on:One piece of the FF170Hp nitrocellulose membraneOne piece of the FF80HP nitrocellulose membrane.
?. Glue the absorbant pad with tape at the top edge top of the 4 pieces of nitrocellulose. the dimensions of the absorbant pad are 0.8 cm x 1.2 cm.
?. Deposit 0,33 ul of streptavidin solution with a micropipette. Repeat this procedure 3 times, letting the drop dry in between each time.The procedure must be done in the 4 pieces of nitrocellulose.
?. Let the pieces dry overnight at 4ºC in a desiccator.
?. Perform a thin-layer chromatography with PBS-Tween as the solvent: pour 15 ml of the solvent and position the pieces with the absorbant touching the bottom of the container. Let the solvent flow trough the nitrocellulose for 15 min.The assay can be done individually or with the 4 pieces of nitrocellulose at one time.
?. Let it dry at room temperature for 40 min
?. Reveal with  Ponceau 0.1% within acetic acid 5% by immersing the nitrocellulose piece into the solution for a few seconds.
?. Destain in dH2O in agitation for 2 min.
TITLE: Algae strain maintenance (Chlamydomonas reinhardtii)
AUTHORS: Joao Vitor Molino

[STEPS]
?. [Plate preparation]
Always follow aseptic techniquesAutoclavate freshly prepared TAP media with   with a magnetic bar insideMix the flask using a magnetic stirrer to evenly dissolve the agar in the solutionFor antibiotic containing plates, cool down the flask temperarute until    -    or until it is possible to hold it without hurting the skin. Add antibiotics in the desired amount, and mix using a magnetic stirrer to evenly dissolve the antibioticPour the molten media in sterile petri dishs. (Keep volume constant among plates).Wait for media to solidifyFor antibiotic containing plates, cool down the flask temperarute until   -   or until it is possible to hold it without hurting the skin. Add antibiotics in the desired amount, and mix using a magnetic stirrer to evenly dissolve the antibioticPour the molten media in sterile petri dishs. (Keep volume constant among plates).Wait for media to solidifyStock at -  until use. (Storage for 2 months was used without noticeable difference)
[bacteriological agar]
55 °C
60 °C
4 °C
8 °C
?. [Innoculation]
Keep agar plates in the bench until they reach From a plate or liquid culture contaning the algae sample, collect a portion and applied it to the fresh plates. From a dense culture ()  to   suffice. From a agar plate, scratch the lawn with the innoculation loop until the loop is full.Wrap the plate with parafilm, and place it at  , 60-80 µmols de photons/m2s for   (5days).
0 Room temperature
50 µl
100 µl
25 °C
?. [Storing]
Place plates with the newly grown cells at a dark enviroment and After .  (Dark is needed to avoid overgrown of the cells, due to photosynthesis. Media contains acetate which keeps the cells viable over a month).
0 Room temperature
?. [Re-stocking]
After  (1 month), cells should be pass to a new plate. Longer periods are possible, but increase the chance of losing the strain.
TITLE: Peptostreptococcal Protein L and Protein LG sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between Staphylococcal protein-L (SpL) and protein-LG with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA was used to study the interactions between Staphylococcal protein-L (SpL) and protein-LG with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpL in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpLG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark.
?. The reaction was stopped with 50 µL of a 3M H2SO4 solution. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off point was set to 0.32.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Total genomic DNA was extracted from leaf tissue to amplify and sequence <span style="display: inline !important; float: none; background-color: transparent; color: #000000; font-family: 'Open Sans',sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: 400; letter-spacing: normal; line-height: 16px; orphans: 2; text-align: left; text-decoration: none; text-indent: 0px; text-transform: none; -webkit-text-stroke-width: 0px; white-space: normal; word-spacing: 0px;">individually five</span> specific target loci from nuclear and chloroplast genome.</p>

[STEPS]
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TITLE: Human and Mouse Islet Single-cell Dispersion for Patch-clamp and Imaging
AUTHORS: Aliya Spigelman, Austin B, Xiaoqing Dai, Amanda Gomes, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Detailed protocol for dispersing human and mouse islets into single cells for patch clamp and imaging experiments</div></div>

[STEPS]
?. Pick islets into 15 mL falcon tube with as little media as possible.
?. Add 1ml of Gibco cell dissociation buffer (cat # 13150-016).
[Cell dissociation buffer]
?. Incubate in 37°C water bath for 10 minutes.
37 °C
?. Pipette islets up and down with 1 ml pipette to produce single cells
?. Add 9 ml of human islet culture media.
[Human islet culture media]
?. Centrifuge @200 RCF for 3 minutes.
?. Aspirate supernatant and resuspend pelleted cells in human islet media (~200 ul/dish)
?. Plate ~200 ul drop of suspended cells in centre of each tissue culture treated 35 mm dish.
?. Culture @ 37°C in 5% CO2 incubator
[5% CO2]
?. After 4 hours add 2 mL of human islet culture media.
[Human islet culture media]
?. Transfer islets into 10cm petri dish (non tissue culture treated), adding human islet culture media if needed.Human islet culture media:  AB1500ml DMEM (5mM glucose)
Gibco 11885
250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep
Gibco 15140-122
AB1500ml DMEM (5mM glucose)
Gibco 11885
250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep
Gibco 15140-122
?. Under a stereomicroscope, hand pick islets into a 2nd 35-mm non tissue culture treated dish with Human islet culture media. The number of islets will depend on how many dishes are required. Our group uses 10 islets/dish as a maximum final density (ie 60 islets for 6 dishes).
?. Repeat this into a subsequent 35-mm petri dishes until islets reach close to 100% purity.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Even though, progesterone is a hormone naturally produced by ovary, but whether the external progesterone is as beneficial as the natural for female? What alteration occurs due to their prolonged use in different organs of female reproductive tract? We aimed to observe the histopathological changes brought by the long term use of Depot-medroxyprogesterone acetate (DMPA), which is the highly popular and promoted contraceptive in developing countries, in reproductive tract of female rat particularly in ovary and Uterine tube. There were significant changes in the histo-morphology of ovary and uterine tube of experimental group rat compared to the control group. Histological changes such as follicular atresia, medullary hemorrhage and atrophy of uterine tube epithelium was seen in experimental group rat. In conclusion, long- term use of exogenous progesterone, such as DMPA for contraception has adverse impact in Female which outweigh the merits. Instead of promoting these hormonal product, which in long period attenuate the health of female, other non-hormonal methods of contraception should be publicized along with their efficacy.</p>

[BEFORE_START]
<p>For this study, only healthy female rats of appropriate weight were taken. </p>
<p>Heath of the rat was assessed by observing thier appetite, movement and external appearance.</p>
<p> </p>

[GUIDELINES]
<p>Safe approach to animal must be assured. In this study following issues were considered for better result:</p>
<ol>
<li>Animals were housed in a well equiped and sterilized animal lab.</li>
<li>Sufficient space for the mobility of animal was provided.</li>
<li>Healthy diet was supplimented.</li>
<li>Animals were sacrified according to the institutional ethical board guidelines. </li>
</ol>

[STEPS]
?. 
?.
TITLE: KAPP-Sen TMC: Term Placental Tissue Collection
AUTHORS: Sonja Suvakov, Elizabeth Ann L. Enninga, Bridget A. Schley, Janelle Santos, Vesna D. Garovic

[DESCRIPTION]
The placenta samples are obtained within 15 minutes after delivery. As soon as the placenta is delivered it is placed in the box at room temperature and brought to the room where the samples are collected and labelled. Approximate time needed to complete the collection is 30 minutes.

[STEPS]
1. 
 


2. Maternal side samples were collected within 3-5 cm range from the place where UC would be projected, cut into ~1 cm cubes and placed either in the cold MACS or cassettes with 10% buffered formalin.
3. Dissection of the fetal side of the placenta were made within 3-5 cm range from the UC and cut into ~1 cm cubes and placed either in the cold MACS or cassettes with 10% buffered formalin.
4. For the umbilical cord we cut the part that was closest to the baby. A total of ~3cm was cut and divided into the smaller pieces. The samples were immediately stored either in the cold MACS or cassettes with 10% buffered formalin.
5. Chorion was sampled from the fetal side per the instructions of Dr. Santos. It may contain the pieces of amniotic membrane as it was difficult to remove one from the other. A 3 cm cube was removed and divided into three smaller pieces which were stored as described in the previous steps. Note: fetal side=chorion
6. For the amniotic membrane we collected the very distant section that was wrapped around baby (edge). Same storage/transfer to the lab.
7. Upon arriving to the lab, all samples were prepared for shipping. Tissue stored in the cold MACS was transferred to new tubes wither with fresh, cold MACS (for scRNAseq) or flash frozen (for snRNAseq). Cassettes initially placed in the formalin jar were transferred to new formalin jars.