eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-variant-numeric: inherit; font-weight: bold; font-stretch: inherit; font-size: 14px; line-height: inherit; font-family: Raleway, sans-serif; vertical-align: baseline; position: relative !important; top: auto !important; left: auto !important; bottom: auto !important; right: auto !important; color: #000339; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff;">SpinSmart Plasmid Miniprep kits</strong><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;"> are designed to rapidly purify plasmid DNA from bacterial cultures.</span></p>
<p> </p>
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">Please follow one of the protocols below, depending on whether you are purifying h<span style="color: #000000; font-family: Arial; font-size: 13px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: pre-wrap; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">igh-copy plasmid or low-copy plasmid/P1 constructs/cosmids.</span></span></p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.</p>
<p> </p>
<p>As a minimum, prepare 12 sample bottles for cells infected with phage and control phage lysate, under 2 different media treatments, with triplcates of each unique Phage/Treatment combination.</p>

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

[GUIDELINES]
<p>-Protocol can be used for Beta Amyloid products that list “IHC” as an application on the datasheet (ex 4G8, 6E10, etc)</p>
<p> </p>
<p>-Use with Ultra Streptavidin Detection Kit (<a href="https://antibody.biolegend.com/datasheet.php?UpProd=&amp;catalogno=SIG-32250" target="_blank">SIG-32250)</a> or (<a href="https://antibody.biolegend.com/datasheet.php?UpProd=&amp;catalogno=SIG-32248" target="_blank">SIG-32248)</a>. All steps should be done in a humidity chamber such as <a href="https://antibody.biolegend.com/datasheet.php?UpProd=&amp;catalogno=SIG-31031" target="_blank">SIG-31031</a></p>

[STEPS]
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TITLE: Complete Medium or Complete Medium Xylose  (from Leach, Lang and Yoder 1982)
AUTHORS: Megan Mcdonald

[DESCRIPTION]
For the growth and maintenance of Cochliobolus carbonum and Cochliobolus victoriae

[STEPS]
SECTION: Make 100x Salt Solutions A and B
2. 100X Salt Solution A

10g Ca(NaO3)2 * 4 H2O
100 mL ddH2O

Autoclave

Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Make 100x Salt Solutions A and B
3. 100X Salt Solution B

2 g  KH2PO4 * 7 H2O
1.5g NaCl

100 mL H2O
pH 5.3

Autoclave

Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Make Complete Medium (CM) or Complete Medium Xylose (CMX)
4. Complete Medium Base

10 g  glucose OR xylose (substitute glucose for xylose for CMX medium)
1 g Yeast Extract
1 g Casein
20g Agar
10 mL Salt A
10 mL Salt B

Make up to 1000 mL with ddH2O and Autoclave

After autoclaving add:
1 mL  sterilised micronutrient solution


Citation:
Leach, J., Lang, B. R. & Yoder, O. C. Microbiology128, 1719-1729, doi:https://doi.org/10.1099/00221287-128-8-1719 (1982).
SECTION: Make Micronutrients Solution
1. 9 mg  H3BO3
58.5 mg CuSO4 *5H2O
1.95 mg KI (Potassium Iodine)
9 mg MnSO4
7.6 mg NaMoO4
822 mg ZnSO4 * 6 H2O
139.8 mg FeCl3 * 6H2O

in 300 mL ddH2O and filter sterilise

Citation:
Heterokaryosis and Parasexuality in the Fungus Ascochyta Imperfecta Author(s): K. E. Sanderson and A. M. Srb Source: American Journal of Botany , Jan., 1965, Vol. 52, No. 1 (Jan., 1965), pp. 72-81 Published by: Wiley Stable URL: https://www.jstor.org/stable/2439977

SECTION: Complete Medium for Sporulation
5. Complete Medium Base

0.5 g  glucose OR xylose (substitute glucose for xylose for CMX medium)
20 g Sorbose
1 g Yeast Extract
1 g Casein
20g Agar

10 mL Salt A
10 mL Salt B

Make up to 1000 mL with ddH2O and Autoclave

After autoclaving add:
1 mL  sterilised micronutrient solution
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the recipe for apple-agar plates which can be used for embryo collection. Alternatively, grape juice can be used instead of apple juice.</p>

[GUIDELINES]
<p>This protocol describes how to make sufficient apple-agar for approximatley 10x 90mm Petri dish plates. </p>
<p>Once the apple-agar food is decanted, the plates should be left, covered by lids, at room temperaure until the food has set and cooled. The plates should then be sealed in an air tight bag and stored at 4°C for a maximum of ~ 6 weeks before use.</p>
<p>The plates should be brought to room temperature before being added to a population cage.</p>

[STEPS]
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TITLE: RNA to cDNA and RT-PCR protocol
AUTHORS: Heather Robeson, Jing Jin, Mohammed S. Orloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Used applied biosystems high-capacity RNA-to-cDNA kit and fast SYBR green master mix protocol</div></div>

[STEPS]
?. [RNA to cDNA reverse transcription]
Prepare the RT reaction mix2X buffer mix :20X RT enzyme Mix: RNA sample: Nuclease-free water: quantity sufficient to Total per reaction:
10 µl
1 µl
9 µl
20 µl
20 µl
?. [RNA to cDNA reverse transcription]
Prepare the reverse transcription reactions:Aliquot   RT reaction mix into each wellSeal the plates and centrifuge to spin down the contents and remove air bubblesStore them on ice
20 µl
?. [RNA to cDNA reverse transcription]
Perform reverse transcription:Step1:Temp.  , time: 60 minsStep2:Temp.  , time: 5 minsStep3:Temp.  , time: unlimitedStore the cDNA:Short-term: Long-term: store at −25°C to −15°C
37 °C
95 °C
4 °C
?. [Prepare PCR reaction plate]
For one reaction:Fast SYBR® Green Master Mix (2✕) : Forward and Reverse Primers : variablecDNA template + RNase-free water: variableTotal: Mix by gentle inversion, then centrifuge the tube to spin down the contents and eliminate any air bubbles
10 µl
20 µl
?. [Prepare PCR reaction plate]
Transfer the appropriate volume of each reaction to 384-well reaction plate
?. [Run the PCR reaction plate]
Instrument: 7900HTAmpliTaq Fast DNA Polymerase, UP Activation :Temp.  , time 10 mins, Cycles HOLDDenature :Temp.  , time 1secondTemp.  , time 20 secondsAnneal/extend:Temp. , time 15 secondsTemp.  , time 15 secondsTemp.  , time 15 secondsReaction volume: MicroAmp Optical 384-Well Reaction Plate
95 °C
95 °C
60 °C
95 °C
60 °C
95 °C
20 µl
?. [Run the PCR reaction plate]
Analyze the data
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Use for solid phase culture and chemical selection of <em>Pseudo-nitzschia spp</em> and other recalcitrant diatoms</p>

[GUIDELINES]
<p>Reagents:</p>
<ul>
<li>0.2 μm filtered seawater (FSW)</li>
<li>L1 Medium Kit (ncma.bigelow.org)</li>
<li>SeaPrep™ Agarose (Lonza Cat#: 50302, 25 gm)</li>
</ul>

[STEPS]
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TITLE: DNA extraction (BOMB)
AUTHORS: Yin-Tse Huang, Tsu-Chun Hung

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 0.5 mm beads to 2mL screw tube
  

SECTION: Sample Collection
2. Add200 µL of 1 mm beads to 2mL screw tube
 
SECTION: Sample Collection
3. Add870 µL Lysis master mix to 2mL screw tube. The final look:
 
 

SECTION: Sample Collection
4. Collect 20-50 mg of sample to 2mL screw tube

 

SECTION: Sample crush
5. Put the 2mL screw tube in mixmill for sample crush, at 3200 rpm  4 min 

 

SECTION: Centrifugation
6. Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 3 min, 25 °C 
SECTION: DNA purification
7. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
0.20. Add 100 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 
  
 


SECTION: DNA purification
14. Add 300-500 µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate
  
 


SECTION: DNA purification
9. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate
  

SECTION: DNA purification
10. Add 300 µL of 80% ethanol to the 3rd well of 96 deep well plate
 
SECTION: DNA purification
11. Add 300 µL of 95% ethanol to the 4th well of 96 deep well plate
    

SECTION: DNA purification
13. Add 100 µL of DEPC-treated water to the 6th well of 96 deep well plate 
 

SECTION: DNA purification
15. Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol

SECTION: DNA purification
16. After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.
  

SECTION: DNA purification
12. Add 300 µL of DDW to the 5th well of 96 deep well plate
  

SECTION: DNA purification
17. Collect 100 µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments
  

SECTION: DNA purification
8. Add 50 µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate
TITLE: Preparation of primary rat cortical neuron and astrocyte co-culture 
AUTHORS: mineechoi

[DESCRIPTION]
This protocol describes how to prepare primary rat cortical neuron and astrocyte co-culture. 

[STEPS]
1. 1-3 days postpartum Sprague Dawley rats (University College London breeding colony) are used. 
Experimental procedures are performed according to the United Kingdom Animal (Scientific Procedures) Act of 1986. 
2. Rat cortices are placed in an ice-cold Dissecting buffer (described in Materials).
3. Wash five times with Washing buffer (described in Materials).
4. Tissues are digested with a Disgesting buffer (described in Materials) for 15 min

5. Digested tissues are neutralized with a dissecting buffer. 
6. Washing twice with a Washing buffer, 
7. Dissociate with a Washing buffer supplemented with DNAse.
8. Dissociated pellets are collected in Neurobasal completed medium.
9. Approximately 600,000 cells are plated on  Poly-D-Lysin (PDL) coated coverslips and 200,000 cells for 8-well ibidi chambers (PDL coated). 

10. The cultures are maintained at 37 °C (5 % volume CO2), and the media are changed every 4-5 days. 
11. Cells can be used at 12-16 days.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
One-step growth curves are used to make determinations about the life cycle of a virus on a particular host.  By following a virus infection during one life cycle phase of host a growth curve can be constructed and the burst size can be calculated.

[BEFORE_START]
<p>Before performing a one-step experiment, you need to determine when the host is growing exponentially.  You must also determine the titer of the phage lysate that will be used for the experiment.  These are described in the protocol.</p>

[GUIDELINES]
Note: One-step growth experiment instructions are for MOI=0.1.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<p>This protocol was developed at this laboratory but had not been previously published.</p>
<p>The protocol aims explicitly to amplify hepatitis A virus (HAV) strains and not other virus species. The assay targets the 5' untranslated region and is designed as a qualitative test for investigating suspected human cases of HAV infection.</p>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<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 familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
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TITLE: Reverse transcription with SuperScript VI VILO
AUTHORS: Molly Moynihan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is from Invitrogen.</div><div class = "text-block"><span style = ":;"> </span><a href="https://www.thermofisher.com/order/catalog/product/11766050#/11766050" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.thermofisher.com/order/catalog/product/11766050#/11766050</span></a></div></div>

[STEPS]
?. [DNase Digestion]
Check for DNA contamination in RNA by performing PCR with universal primers (16S or 18S) (see PCR protocol). Doing the PCR first will help avoid unnecessary DNase use and wasting RNA.ORProceed directly to DNase treatment without initial check.
?. [DNase Digestion]
Gently mix and incubate samples at  for . Briefly spin down and place on ice.
37 °C
?. [Reverse Transcription]
Prepare reverse transcription reactions on ice. Add the following to the 10µl DNase reactions above, or if no DNase treatment was performed, prepare the following in empty PCR tubes. Each sample should have 1x RT and 1x no RT reaction. RT and no RT reactions should use the same amount of template RNA.RT:  Superscript IV Master Mix OR  up to  (aim for 1000 - 1500ng RNA) nuclease free water to final volume of 20µl No RT controls:  Superscript IV no RT control  OR  up to  (aim for 1000 - 1500ng RNA) nuclease free water to final volume of 20µl
4 µl
[DNase reaction]
[of template RNA]
4 µl
[DNase reaction]
[of template RNA]
?. [DNase Digestion]
For samples with DNA contaminationOn ice, prepare 2x DNase reactions for each sample. One reaction will be used for reverse transcription (RT) and the other for the no reverse transcription control (no RT). The no RT control is used to check for DNA contamination. RT and no RT reactions should use the same amount of template RNA.up to  (aim for 1000 - 1500ng RNA)nuclease free water to final volume of 10µl
[10x ezDNase Buffer]
[ezDNase enzyme]
[template RNA]
?. [Reverse Transcription]
Incubate RT and no RT reactions following the settings below (7.1). This can be performed in a thermocycler.
?. [Reverse Transcription]
cDNA can be used directly in PCR or qPCR. Store at  for short term storage or  for long term storage.
-20 °C
-80 °C
?. [Reverse Transcription]
After closing PCR tubes, briefly spin down samples.
?. [Reverse Transcription]
Incubate at  for  (anneal primers) for  (reverse transcription) for   (enzyme inactivation)
25 °C
50 °C
85 °C
?. [DNase Digestion]
Tip: After RT is performed (Step 7), if DNA contamination is observed in the no RT control and DNase treatment was performed, try repeating the protocol with 2x DNase treatments. After the initial incubation at 37°C, add 1µl 10x ezDNase Buffer and 1µl ezDNase enzyme. Incubate again at 37°C for 5 minutes.
TITLE: UC Davis - Massons Trichrome
AUTHORS: Jennifer Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Massons trichrome staining is used for detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red.</div><div class = "text-block"><span style = "font-weight:bold;">Modified from:</span><span> Massons Trichrome UC Davis Clinical Pathology lab</span></div></div>

[STEPS]
?. Hydrate to water.
?. Place in Bouins for one hour in 55 degree oven or overnight at room temperature if needed.
?. Wash well in running water until yellow is gone.
?. Stain in Weigerts Hematoxylin for 10-20 minutes.
?. Wash well in running water for 10 minutes. Rinse distilled water.
?. Place slides in Biebrich Scarlet-Acid Fuchsin for 5 minutes.
?. Rinse in distilled water.
?. Place slides in the Phosphomolybdic-Phosphotungstic Acid Solution in a plastic coplin jar and put
in the Bio Rad for 25 seconds using the total time setting. Let stand 30 seconds.
?. Place slides in Aniline Blue solution for 10 minutes.
?. Rinse in distilled water.
?. Place in 1% Acetic acid for 3 minutes if desired.
?. Dehydrate and coverslip.
TITLE: SM buffer
AUTHORS: Frej   Larsen

[DESCRIPTION]
SM buffer is used for diluting and storing bacteriophages.

SM buffer contains:
200 mM NaCl2
10 mM MgSO4
50 mM Tris-HCl, pH 7.5

For storage at -20 or -80° C, 15% glycerol can be added before autoclaving.


The buffer can keep at room temperature for several months. 

[GUIDELINES]
This recipe is for 1 liter of buffer. Make sure to recalculate the measurements if you are making a smaller or larger volume.

Ensure that your MgSO4 is a monohydrate before weighing. If not, recalculate amount required to reach correct concentration.

[STEPS]
1. Weigh off 11.7 gNaCl2 and 1.4 gMgSO4 and add to a clean 1L blue cap flask or other autoclave safe container.

2. Measure 50 mL Tris-HCl and add to the flask. 

3. Measure 950 mL demineralized water and add to the flask.

4. Screw the cap on tight and mix thoroughly for 30 s.

5. Loosen the cap and autoclave.
6. If there are still undissolved particles, mix until dissolved. Then let it cool at room temperature.
TITLE: Intracardiac perfusion and rat brain fixation for immunohistochemistry
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for rat brain processing to perform parallel histological analysis in one hemisphere and high dimensional flow cytometry in the other.   

[STEPS]
SECTION: 1. Animal sacrifice
1. Before starting, fill the tube of a peristaltic pump with ice-cold 1X PB solution 
SECTION: 1. Animal sacrifice
2.  Deeply anesthetize the animal. Check for no reflexes and slower breathing
SECTION: 1. Animal sacrifice
3. Cut the animal's thorax and perfuse the animal through the left ventricle with ice-cold 1X PB solution, after a small cut of the right atrium to allow fluid flux  
SECTION: 1. Animal sacrifice
4. Remove brains from the skull and divide the two hemispheres into two halves along the medial sagittal line (one hemisphere is freshly dissected and processed for high dimensional flow cytometry, and the other one is processed for histological analysis as follow)
SECTION: 2. Postfixation
5. For histological analyses, postfix one brain half in 40 ml formaldehyde solution 4% in PB buffered for 72h, at 4°C (replace every 24 hours with fresh fixative solution)
SECTION: 3. Brain processing for cryostat sectioning
6. Rinse three times in PB and immerse in 30% sucrose + 10% glycerol solution at 4 °C until sinking for cryoprotection
SECTION: 3. Brain processing for cryostat sectioning
7. Include brains with OCT 
SECTION: 3. Brain processing for cryostat sectioning
7.1. Freeze in 2-methylbutane, cooled into liquid nitrogen, and store at -80 °C
SECTION: 4. Cryostat sectioning
8. Perform sectioning with a cryostat at 30-μm-thickness  
SECTION: 4. Cryostat sectioning
8.1. Store sections at 4°C in 0.1 M PB containing 0.02% sodium azide before being further processed for immunohistochemical staining
TITLE: Study Procedures (Part 6 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 6 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
?. [Random Assignment]
Participants who sign the informed consent and meet the eligibility criteria will be randomly assigned in a 2:1 ratio to either the experimental or control group. A central automated randomization system will be used for treatment assignment and to create a unique identifier for each new study participant. Random assignment will be stratified according to site.
?. [Blinding]
Blinding will be maintained throughout the study for all study participants and study personnel, except the pharmacists.
?. [Unblinding]
Unblinding before the study is completed will occur only if a participant’s well-being is threatened and the investigator believes unblinding is necessary to protect the participant.
?. [Unblinding]
Before treatment assignment for an individual participant is unblinded, the investigator must confer with the study sponsor and the medical monitor. The site investigator will notify the protocol chairs of the unblinding event, and the medical monitor will notify the study management team (SMT).
?. [Unblinding]
The emergency unblinding will be recorded and reported to the DSMB. A full account of the event will be recorded, including the date and time of the emergency, the reason for the decision to unblind, and the names of the medical monitor and others who were notified of the emergency. During site visits, the site monitor must verify that the medical monitor was notified and that a written account was completed. The reasons for unblinding of a participant’s treatment will be included in the final study report.
?. [Unblinding]
Sponsor approval is required for unblinding the treatment of an individual participant or subgroups of participants for unplanned interim analyses to support DSMB reviews and final analysis.
?. [Visit Windows]
Scheduled VisitsAll scheduled study visits in Appendix 1 must occur within the time limits specified below. Visits that occur outside of the specified windows will be considered protocol deviations.Visit -1         Up to 2-4 weeks before visit 0Visit 0          No windowVisit 1          (± 3 days)Visits 2-7     (± 7 days)Visits 8-11   (± 14 days)