eamag/protocols-with-steps huggingface dataset:
TITLE: Electroporation of Cas9 protein into human pluripotent stem cells
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the electroporation of Cas9 protein into human pluripotent stem cells.</div></div>

[STEPS]
?. Add  to a sterile 1.5 ml tube. Add . Then add . Pipet up and down to mix. Let it sit at  for . This is enough for 2 transfections (== one 6 well).
[buffer R ]
[purified Cas9 protein (2mg/ml)]
[sgRNA]
0 Room temperature
?. While waiting for the Cas9 to bind to sgRNA, individualize cells with Accutase. Neutralize Accutase with 5x volume E8 with Rock inhibitor.
?. Count cells. You will need 2x105 for each transfection.
?. Spin down cells. Let it sit for a while so all the residue media can go down to the bottom of the tube. If the residue media is too much, take it out with a P200 pipet.
?. Resuspend cells to a concentration of 2x105 per 5 μl (ie 4x107 per ml) using buffer R.
You don’t have to take all the residue media off but you will need to take into account the volume of residue media so you are not too much off.
?. Prepare a 24 well matrigel coated plate. Add - to the wells you will use. Add HAS (1:2500) to each well. Each transfection goes into one well.
0.5 mL
[E8+ rock inhibitor (1:1000)]
?. Wipe the Neon pipet station with EtOH and place it inside the hood.
?. Add  to the neon tube. Place the tube inside the station. You should feel a click before the tube is securely seated in the station.
[electrolytic buffer (buffer E)]
?. Use program 13 from the optimization tab for electroporation parameter. Program 9 should also work.
?. When everything is ready, mix - with the Cas9+RNA containing R buffer. The final volume should be in the range of -.
10 µl
[of resuspended cells]
21 µl
22 µl
?. Take up a neon tip, pipet  and electroporate with program 13.
[cell protein mix]
It is important to pipet slowly to avoid air bubble formation. It is also important to insert the pipet slowly into the station, especially during the end of the insertion when you will feel a click. I normally help the pipet down slowly during the clicking so there is no sudden movement of the tip, which might create tiny air bubbles.
?. If you see air bubble in the tip, take it out, push everything out of the tip and repipet the mixture.
?. If you see sparking during the electroporation, your efficiency will reduce significantly.
?. Once electroporation is complete, push everything into one well of a 24 well plate. Do not pipet up and down with Neon tip.
?. Repeat the same procedure with the same tip and the left over cell mixture. This is just a replicate.
?. Disperse cells evenly in the well and place cells in a low O2 incubator.
?. Put electroporated cells into low oxygen incubator for 2 days (Rm 329, key can be found at Melissa’s desk).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>I and my team used this assay between 2008-2015; we dubbed it the "Harvala assay". It produces an amplicon that includes the 3' end of VP3 and the 5' end of VP1, spanning the junction.</p>
<p><em>In silico </em>sequence alignments indicated the highly degenerate olignucleotides could theoretically detect af least HPeV 1-7, 17 and 18.</p>

[GUIDELINES]
<ul>
<li>This protocol assumes the user is familiar with working in a laboratory, with PCR, agarose gel electrophoresis, personal protective equipment, and use of a thermocycler</li>
<li>This protocol should be re-evaluated if being used with different reagents, if the oligonucleotide sequences are changed or if the cycling conditions are altered </li>
</ul>

[STEPS]
?. 
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?.
TITLE: Isolation of fungi associated with ambrosia beetles with pre-oral mycangia
AUTHORS: Kyra Lynn, Irene Barnes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to extract fungi from ambrosia beetles that have pre-oral mycangia.</div><div class = "text-block">Note: This protocol is specific for ambrosia beetles with pre-oral mycangia and may not be useful for beetles with different mycangia types.</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]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Modified from Harrison et al., 1980. A broad spectrum artifical seawater medium for coastal and open ocean phytoplankton. The final salt concentration is ~34 ppt.</p>
<p> </p>

[BEFORE_START]
<p>Acid washed all glassware with 10% HCl and rinse well (3-5 times with deionized or ultrapure water depending on downstream application). Prepare L1 medium components, L1 trace element solution and f/2 vitamin mix ahead of time.</p>

[GUIDELINES]
<p>Prepare solutions separately, autoclave (or filter sterilize as needed), and then mix to prevent precipitation of salts. When autoclaving, use a bottle at least twice the size of the needed volume (e.g. use a 1 L bottle to autoclave 500 mL). Solutions should sit for 24-48 hr after autoclaving to allow for gaseous exchanges. N, P, Si, and trace element components are from the Bigelow NCMAM L1 medium; Vitamin components are from the Bigelow NCMAM f/2 medium. Trace metal and vitamin solutions can be made ahead of time and aliquots stored frozen.</p>

[STEPS]
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?.
TITLE: Double Digestion and Dephosphorylation of Plasmid
AUTHORS: iGEM Dusseldorf

[STEPS]
?. Mix the following components gently:  AB1Componentsng/µL2Plasmid DNA Tube X3ComponentVolume (µL)4Sterile MilliQ WaterFill up to 30.0 µl510x FastDigest Buffer3.06Plasmid DNATotal ng of DNA/Plasmid DNA concentration ng/µl7FastDigest Enzyme 11.08FastDigest Enzyme 21.09FastAP Alkaline   Phosphatase1.010Total Volume30.011Total ng of DNA1000.0
AB1Componentsng/µL2Plasmid DNA Tube X3ComponentVolume (µL)4Sterile MilliQ WaterFill up to 30.0 µl510x FastDigest Buffer3.06Plasmid DNATotal ng of DNA/Plasmid DNA concentration ng/µl7FastDigest Enzyme 11.08FastDigest Enzyme 21.09FastAP Alkaline   Phosphatase1.010Total Volume30.011Total ng of DNA1000.0
?. Incubate at enzyme suitable conditions  -   and temperature
Find out suitable conditions on website of particular science company
?. Inactivate the enzymes at suitable conditions  and temperature
Find out suitable conditions on website of particular science company
?. Run gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
?. Cut out digested band (~150 mg) and transfer to pre-weighed tube. Check weight.
?. Purify the band
TITLE: U Michigan - Retinal Microstructure Imaging OCT
AUTHORS: David A. Antonetti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">A spectral domain ophthalmic imaging system based on the technology of optical coherence tomography (OCT) will be used to acquire the microstructure image of retina in live animals. Like the ultrasound, but uses light waves instead of sound waves, the light backscattered from within a sample is processed to develop a high-resolution, depth-resolved image suitable for analyzing internal microstructure in vivo.  </div></div>

[STEPS]
?. The eyes of conscious animals are dilated by first applying a small drops (~10 µl) of tropicamide (1%) followed by a small drop of phenylephrine (2.5%) 2 to 3 minutes later
?. Animal is then sedated with ketamine and xylazine
?. After sedation, the corneas are kept moist with the wetting agents (Systane)
?. Animal is then placed on a platform enabling three-dimensional fine movements
?. The camera lens is adjusted to aim at the eye to acquire images without physical contact
?. Volume or radial scan (1.4 x 1.4 mm) is obtained from each eye with a Bioptigen Envisu R2200 preclinical spectral domain optical coherence tomography (SD-OCT) system
?. After imaging procedure, the animal is allowed to recover from the anesthesia
?. Bioptigen Diver software is used to analyze retinal structure from the image obtained
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Clean the benches and all the material that will be used with alcohol 70. </p>
<p>Use tips with filter. </p>
<p>Add 4 volumes of 100% ethanol to the RPE buffer.</p>
<p>To lyse the cells, you can use β-mercaptoethanol or 2 M dithiothreitol. For each 1ml of the RLT buffer, add 10 μl of β-mercaptoethanol. For each 1ml of the RLT buffer, add 20 μl of 2 M dithiothreitol.</p>
<p>You must use a maximum of 1 x 10<sup>7 </sup>cells.</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time PCR method for the detection of Molluscum contagiosum virus from human samples. This assay targets the MC021L gene and detects both subtype 1 and 2 of the virus.</p>

[BEFORE_START]
<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 familar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
?. 
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?.
TITLE: Conjugation of peptide fragment 579-601 of HIV-gp41 or fragments 308-331 or 421-438 of the HIV-gp120 with keyhole limpet hemocyanin (KLH).
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Chemical synthesis facilitates the generation of peptides which are very difficult to express in bacteria, peptide/protein backbone modification,  the incorporation of unnatural amino acids, and the production or synthesis of D-proteins. </div><div class = "text-block">The C-terminal cysteine can be added to the amino acidic sequences of HIV peptides (fragment 579-601 of the HIV-gp41 and fragments 308-331 or 421-438 of the HIV-gp120). These peptide fragments were dimerized by cysteine oxidation with dimethyl-sulfoxide [1] to faciliutate their conjugation to keyhole limpet hemocyanin that acts as a carrier protein.  </div><div class = "text-block">Reference:</div><div class = "text-block">1. Tam JP, Wu CR, Liu w, Zhang JW (1991) Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications. J Am Chem Soc 113: 66576662. </div></div>

[STEPS]
?. These peptide fragment (579-601 from HIV-gp41) is dimerized by cysteine oxidation with dimethyl-sulfoxide.  The HIV peptide is dissolved in 5% acetic acid to a final concentration of 5.1 mg/ml.
?. The  pH of the medium is adjusted to 6 with 1 M (NH4)2CO3.
?. Dimethyl-sulfoxide is added to 20% of the final volume, and after four hours at room temperature (RT), the solute is  extracted.
?. Then, the peptide is dissolved in 3 ml 5% trifluoroacetic acid and precipitated with 35 ml cold ether.
?. The precipitate is dialyzed against 1.2 liters of deionized water, pH 7 at 4°C overnight.
?. Then, 1 mg of keyhole limpet hemocyanin (KLH) is diluted in 2.1 ml 0.1 M borate buffer (1.24 g boric acid, 1.90 g sodium tetraborate, pH 10, in 500 mL deionized water).
?. In a 20 ml glass tube, with a gentle stirring, 1.1 µmol of the HIV synthetic peptide (with C-terminal cysteine added) and 0.22 mL 0.3% glutaraldehyde solution (ACS reagent grade, pH 5.5, Sigma-Aldrich) are slowly mixed at RT and left to stand for 1.50 hrs.
?. When a yellow coloration is observed this indicates that the conjugation process is successful.
?. To blocking  the excess of glutaraldehyde, 0.26 ml of 1 M glycine (Sigma-Aldrich) is added.
?. The mix is left for 32 min at RT.
?. The HIV-hemocynin conjugate is then dialyzed against 1.3 liters 0.1 M of borate buffer, pH 8.4 through the night at 4°C.
?. Then the previous buffer is used to dialyze the preparations for 8 hrs at 4°C.
?. The dialysates is stored at 4°C until further use.
TITLE: Calibrating a pH meter
AUTHORS: PMAT0001 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Step-by-step instructions on calibrating a pH meter</div></div>

[STEPS]
?. [Main steps]
Press “Cal”
?. [Main steps]
Press “✔️”
?. [Main steps]
Dip in pH 4
?. [Main steps]
Press “✔️”
?. [Main steps]
Rinse pH meter
?. [Main steps]
Dip in pH 7
?. [Main steps]
Press “✔️”
?. [Main steps]
Dip into sample; Note percentage purity as well
?. [Important notes]
- When liquid level falls way below the "hole" at the top, replenish with some KCl- After finishing experiments for the day, rinse pH meter, secure top with parafilm and put in KCl (parafilm to the KCl storage as well)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The following protocols is submitted as part of the Cikapundung Research 2017, as can be divided in to 3 submissions:</p>
<p> </p>
<p>1.  Identifying landuse and calculating its alteration in time</p>
<p>2.  Vegetation and zoobentos observation</p>
<p>3.  Water sampling</p>
<p> </p>
<p>OSF folder will be made following this protocols.</p>

[BEFORE_START]
<p>Reminders:</p>
<p>1. make sure to know the route to the safest part of riverbank. Because some locations feature high cliff and difficult sampling position due to near-river housings.</p>
<p>2. make sure to check the equipment conditions: bateries, buttons, cables, probes etc.</p>
<p>3. make sure to take notes of the situation and weather in time of sampling.</p>
<p> </p>
<p>Softwares: Fragstat, Envi, ArcGIS</p>

[GUIDELINES]
<p>The guidelines will be divided to:</p>
<p>1. landuse observation</p>
<p>2. vegetation observation and sampling technique</p>
<p>3. water quality observation and sampling technique</p>

[STEPS]
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TITLE: HV-CTAB-PCI DNA Extraction Protocol
AUTHORS: Vicky Ooi, Lee McMichael, Margaret E. Hunter, Aristide Takoukam Kamla, Janet M. Lanyon

[DESCRIPTION]
A ‘High Volume­­- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol ’ (HV-CTAB-PCI) method was developed.

[STEPS]
1. Clean the working bench with TriGene.
2. Scrape 1 g of faecal material from the outer surface of a faeces and put it into a 15 mL centrifuge tube. 
3. Transfer the faecal material into a mortar and grind the faeces into powder with liquid nitrogen.
4. Add 1 mL of Lysis Buffer 1 (LB1: CTAB 2%, Tris– HCL 100 mM, EDTA 20 mM, NaCl 1.4 M, pH 7.5) to the mortar containing the faecal material to further grind and mix in the buffer with the ground faeces. Transfer the liquid back into the 15 mL tube.
5. Add another 1 mL of LB1 to the mortar to mix in any leftover faecal material on the mortar and transfer the liquid back into the 15 mL tube. Repeat this step once more, and then add 2 mL of LB1 to the 15 mL tube containing the faecal homogenate. Thus, a total of 5 mL of LB1 would be added to the ground faeces.
6. Vortex the faecal homogenate and incubate in a thermomixer for 3 h, with occasional mixing, at 60°C for cell lysis.
7. Centrifuge the sample at 3,150 g (4,000 rpm) for 12 min and pipette 4 mL of the supernatant into a new 15 mL tube.
8. Add 4 mL of phenol: chloroform: isoamyl alcohol (21:20:1) to the supernatant, then gently mix the tube. Centrifuge the sample for 3 min at 3,150 g (4,000 rpm) and pipette 3 mL of the aqueous phase into a new 15 mL tube.
9. Add 330 µL of Lysis Buffer 2 (LB2: CTAB 10%, NaCl 0.5 M, pH 5.5) to the aqueous phase, and leave it to lyse further at 60°C for 4 h.
10. Add 104 uL of protease to the sample and leave it to digest proteins for another 1 h.
11. Add 3434 µL of phenol: chloroform: isoamyl alcohol (21:20:1) to the mixture, and then gently mix the tube well, and centrifuge the sample for 12 min at 3,150 g (4,000 rpm). Then, pipette 3 mL of the aqueous phase into a new 15 mL tube.
12. Add one volume of isopropanol (i.e., 3 mL) to precipitate the DNA overnight at -20°C.
13. Centrifuge the sample for 20 min at 8000 g (5,200 rpm), and then get rid of all the supernatant.
14. Add 400 µL of 70% ethanol to the pellet to wash it. Vortex and then centrifuge the sample at 3,150 g for 12 min and get rid of the supernatant.
15. Dry the pellet at room temperature for 15 min.
16. Resuspend the pellet in 250 µL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8).
17. Store the DNA isolate at -20°C for use within a week or at -80°C for longer-time storage.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>!!! ~~~~~ IN DEVELOPMENT ~~~~~ !!!</p>
<p>This approach uses in-vitro transcription to generate a dinoflagellate-type mRNA (i.e. capped with the dino-SL leader), which is introduced into cells using an RNA transformation reagent. Screening for expression of the protein encoded in the mRNA follows.</p>

[GUIDELINES]
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hk4dnpn.png" data-src="https://s3.amazonaws.com/pr-journal/hk3dnpn.png" data-ofn="mCherry Dino gene.png" /></p>

[STEPS]
?. 
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TITLE: pL0R-LacZ
AUTHORS: Bernardo Pollak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Name</span><span>: pL0R-LacZ</span></div><div class = "text-block"><span style = "font-weight:bold;">Description</span><span>: a L0 receiver vector which can be used for domestication using SapI-mediated restriction ligation or Gibson assembly. Contains lacZ for blue/white colony screening. Requires X-GAL, IPTG optional depending on E. coli strain.</span></div><div class = "text-block">Level 0 </div><div class = "text-block">Overhangs: Special (cuts into BsaI sites)</div><div class = "text-block">Length: 2646</div><div class = "text-block">Antibiotic selection: Spectynomycin</div><div class = "text-block">Other info: pMB1 ori</div><div class = "text-block">Characterisation: ++++ (substantial), works as expected.</div><div class = "text-block">Primers: </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;">U1F</td><td rowspan = "" colspan = "" style ="display : table-cell;">CATTACTCGCATCCATTCTC</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">U1R</td><td rowspan = "" colspan = "" style ="display : table-cell;">GAGACGAGACGAGACAGCCT</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">UXF</td><td rowspan = "" colspan = "" style ="display : table-cell;">CCAGGATACATAGATTACCA</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">UXR</td><td rowspan = "" colspan = "" style ="display : table-cell;">GGTGGAAGGGCTCGGAGTTG</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">L0F</td><td rowspan = "" colspan = "" style ="display : table-cell;">TGGGCTGCCTGTATCGAGTG</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">L0R</td><td rowspan = "" colspan = "" style ="display : table-cell;">GCTGGCCTTTTGCTCACA</td></tr></table><div class = "text-block"> L0 F works great for sequencing.</div><div class = "text-block">Submitter: Bernardo Pollak</div><div class = "text-block">Cloned by: Bernardo Pollak</div><div class = "text-block">Availability: Upon request at the Pollak/Dupont/Federici labs at the moment and will be available soon from Addgene.</div></div>

[STEPS]
?.
TITLE: SPARC Cat - Sham Control Chronic Cat 2 Day 30
AUTHORS: Brett      Hanzlicek, Anna Rietsch, Margot Damaser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a procedure for a sham control chronic cat experiment (Day 30-terminal) for cystotomy (bladder surgery).  The cystotomy is performed without UroMOCA implantation.  The cat is observed daily and imaged on day 0, 14 and 30 to track changes to the bladder and overall cat health in response to the cystotomy.  This protocol includes urodynamics and imaging for Day 30 in the chronic experiments.</div></div>

[STEPS]
?. [Transport Cat]
Transport cat from housing site to surgery site.
?. [Animal Prep and catheter placement]
Animal is anesthetized and abdomen is shaved by the vet team.  The cat is then moved into the surgery room and attached to monitors by the vet team.
?. [Animal Prep and catheter placement]
Drape animal and perform betadine scrub on abdomen and genitals.
?. [Animal Prep and catheter placement]
Put surgilube on 3.5Fr catheter and insert into bladder through the urethra.  Advance the catheter until resistance is met, then pull back 2-3cm.
?. [Animal Prep and catheter placement]
Use gentle suction with syringe to withdraw urine from bladder through the catheter.  Measure the volume and save the urine for urinalysis.
?. [Cystogram - DYNA CT]
Use 1:5 dilution of contrast to saline to visualize bladder.Take a 3D CT image with contrast:saline in the bladder
?. [Cystogram - DYNA CT]
Empty Bladder
?. [Urodynamics/Cystometry 1]
Performed after cat is transferred from Isoflurane to propofol anesthetic.
Fill rate is 2ml saline/minute using syringe pump.
Data is recorded using Labview software.
Pressure is recorded using an external pressure transducer connected to syringe pump on one end and tubing that leads to the catheter on the other end.
?. [Urodynamics/Cystometry 1]
Fill bladder with saline and record pressure using pressure transducer connected to  LabViewFill to  ; Peak pressure around 20 cmH2O
30 mL
?. [Urodynamics/Cystometry 1]
Empty Bladder
?. [Urodynamics/cystometry 2]
Fill bladder with saline at 2ml/min
?. [Urodynamics/cystometry 2]
Fill to  ; End round 2 urodynamics
30 mL
?. [Euthanasia]
Cleveland Clinic animal team euthanizes the animal
?. [Dissection/Histology]
Bladder is removed, examined and placed in formalin for histological analysis
TITLE: Addition of Antibiotic Supplement to Media 
AUTHORS: Mariam  Alkattan

[DESCRIPTION]
The following protocol is for the addition of antibiotic stock solution to molten media. 

[STEPS]
7. Cefotaxime stock solution preparation

0.1. Retrieve molten media from autoclave using heat resistant gloves. Place media in laminar flow hood if one is available. If laminar flow hood is not available, place media next to open flame on lab bench.
 

1. Wait for media to cool to50 °C. Measure temperature using laser infrared thermometer or a stem thermometer. If using stem thermometer, sterile using 70% ethanol and allow to dry right before use. 

2. When media reaches 50 °C , use an adjustable pipette to add appropriate volume of antibiotic stock solution to molten media.

3. Gently swill media in bottle to mix antibiotic.