eamag/protocols-with-steps huggingface dataset:
TITLE: Spot-bleaching of a handful of C. elegans nematode worms
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the spot-bleaching of C. elegans</div></div>

[STEPS]
?. 1. Put a drop (20 to 50 µl) of bleaching solution on the edge of a clean NGM plate seeded with E. coli .
?. 2. Pick several gravid hermaphrodites in the drop. The bleaching solution will kill the contaminants and hermaphrodites but will soak into the plate before the embryos hatch.
?. 3. The next day the L1 larvae will have crawled into the E. coli OP50 lawn. Transfer them to a clean NGM plate seeded with an E. coli OP50 lawn, or cut the patch of dead bacteria/bleach agar.
TITLE: Emotional Control Program
AUTHORS: Sebastião Santos1,2 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Emotions are part of our daily lives, they are the first stage of our learning, they are the first source of knowledge. Emotions and cognition are closely linked. And in this genesis it will be possible to improve and enhance the daily learning that we commonly carry out. The individual, in this perspective, will bring an added value to their performances and in turn to an income they want and expect.</div></div>

[STEPS]
TITLE: OSU TriState SenNet Normal Donor Heart Acceptance Criteria
AUTHORS: Sean D. Stacey, Brenda F. Reader, Lorena Rosas, Victor Peters, Ana L. Mora, Mauricio Rojas

[DESCRIPTION]
This protocol describes the criteria required for the inclusion of human heart specimens from healthy “normal” organ donors in the Comprehensive Transplant Center Human Tissue Biorepository and TriState SenNet Biospecimen Core at The Ohio State University as part of the Cellular Senescence Network Program (SenNet). This protocol is based on the CTC Human Tissue Biorepository's standard operating procedure established for tissue collection for explant and donor samples. We follow routine laboratory specimen processing guidelines to ensure the highest biospecimen quality.

[STEPS]
SECTION: Inclusion Criteria
1. The donor is authorized for research by Lifeline of Ohio, Central Ohio’s organ procurement agency, 18-80 years of age, negative for HCV, HBV, HIV, and has no active COVID-19 infection. The whole heart is procured by Lifeline of Ohio and the biorepository then provides the de-identified heart to the researcher. When processing “normal” donor heart tissue, samples are chosen from the healthiest section of the five target regions of the heart: septum, left and right ventricles, and left and right atrium. The CT scans or chest X-rays are referenced to locate any diseased portions of these regions or to assess the overall health of the donor heart.

To be considered a “normal” donor heart, the donor has no known history of heart failure, no current infection, and limited inflammation.
SECTION: Exclusion Criteria
2. The donor is 17 years of age or younger, HBV, HCV, or HIV positive, or has an active COVID-19 infection.
TITLE: Bogus Experimental Workflow
AUTHORS: Abby   Moore

[DESCRIPTION]
This is a bogus workflow that demos a sequence of resources that cover sample prep, data acquisition and data processing.

[BEFORE_START]
This is what you should know before you start ....

[GUIDELINES]
These are my guidelines ....

[STEPS]
SECTION: Sample prep
1. Bogus Sample Prep Protocol
SECTION: Data acquisition
2. Bogus Data Acquisition Protocol
SECTION: Data data processing
3. Hello World Jupyter Notebook
TITLE: Vector Linearization 
AUTHORS: Felipe FE Edaes

[DESCRIPTION]
Protocol for the linearization of a previously obtained vector.

[GUIDELINES]
Add phosphatase to remove phosphate, which remains from the end of linear vectors, thus preventing cells from relinearizing the vectors.

[STEPS]
SECTION: Measure DNA Concentration of the Plasmid
2. Use 1.0 µL to 1.5 µL of the elution buffer (used to elute the DNA) as blank.

SECTION: Measure DNA Concentration of the Plasmid
3. Measure blank once to confirm the accuracy.
SECTION: Measure DNA Concentration of the Plasmid
4. To measure DNA sample, add  1-1.5 µL 
18. Considering that 1 μg = 1000 ng, divide the measured concentration by the amount required in ng
17.1. If the required amount is 3 μg and the measured sample was 450 ng/μL, the proper calculation would be: 3000/450 = ~6.67 = 7.00 μL
18. 
SECTION: Calculate the amount of Plasmid to be used
5. If 1 µg is 1000 ng, this value should be divided by the amount of DNA measured.
SECTION: Calculate the amount of Plasmid to be used
5.1. If the required amount is 3 µg and the measured sample was 450 ng/μL, the proper calculation should be: 3000/450 = ~6.67 = 7.00 µL

SECTION: Vector Linearization
6. 3 µg Plasmid DNA

SECTION: Vector Linearization
7. 1 µL Enzyme A

SECTION: Vector Linearization
8. 1 µL Enzyme B

SECTION: Vector Linearization
9. 2 µL 10X Buffer

SECTION: Vector Linearization
10. Remaining H2O 20 µL

SECTION: Vector Linearization
11. Incubate the reaction for 180 min at 37 °C, then place it on ice.

SECTION: Equipment
1. 
 

SECTION: Vector Linearization
11.1. Place it on ice and add 0.5 µL phosphatase to the linearized vector.
TITLE: Immunostaining on Paraffin Sections of Fly Heads
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes how to perform immunostaining on paraffin sections of fly heads.

[STEPS]
1. Fix fly heads overnight in 4% formalin.
5. Microwave slides in 10 millimolar (mM) sodium citrate for 15 min. 
Cool 20 min .

Stock: 100 millimolar (mM)  sodium citrate,  pH 6.0
Use at least 1 L of citrate solution in large glass box to avoid drying.





6. Block in PBST (PBS with 0.3% Triton) with 2% dry milk for 30 min 
Stock: 10X PBS
7. Incubate with primary antibody    at Room temperature .

8. Wash 3 x in PBST.
9. Incubate with appropriate biotinylated secondary (for immunohistochemistry) or fluorescent secondary (for immunofluorescence) antibody at 1:200 in PBST + milk for 60 min  at Room temperature . 
For immunohistochemistry incubate in ABC reagent (Vector) for 60 min  at Room temperature .

10. Rinse 3 x PBST.
11. Mount slides with antifading medium for immunofluorescence or dehydrate through ethanol series and xylenes and mount in Permount for immunohistochemistry.
2. Embed in paraffin.
3. Cut 4 micron sections.  Dry at 42 °C  .

4. Deparaffinize and bring through ethanols to water (xylene x 2, 100% ethanol x 4, tap H2O x 2).
TITLE: Callichthyidae (Ostariophysi: Siluriformes) dissection protocol
AUTHORS: Karina Carvalho F. Ferreira, Marcelo Ribeiro de Britto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol shows how to dissect callichthyid fishes without losing importante anatomical information about the relative position and shape of internal organs.</div></div>

[STEPS]
?. Continue the incision in caudal direction to the limit of the celomathic cavity, by passing the pelvic girdle and ending in the point immediately posterior to the pelvic girdle.
?. Remove adipose tissue with the aidof the thin point fórceps in order to better visualize internal organs.
?. Release the organs from the peritoneum using the spacer.
?. Locate the esophagus and make a cross-section on it using the forceps and scissors.
?. Remove the internal organs from the celomathic cavity with the organs without any damage and keepingall the information about shape and relative position of each organ.
?. With the organs outside the celomathic cavity, separate the reproductive organs from the digestive system organs.
?. Remove the connective tissue located between the walls and intertinal loops. Fromthere, the intertinal walls are separated and the digestive tube is distended.
?. Make a incision in the medial portion of the abdomen at the point immediately posterior to the pectoral girdle.
?. Extend the opening from the incision towards the left flank in the ventral view, with the aid of the scissors, bypassing the pectoral girdle towards the dorsal portion of the specimens up to the limit of the celomathic cavity.
?. Lift up the skin and muscles that ventrally with hold internal organs with the aidof the thin point fórceps, and expose the organs.
?. Disconnect the intestine from the anus using forceps and scissors.
TITLE: Human FFPE Liver Pathology Assessment -- University of Minnesota Human TMC
AUTHORS: Oyedele Adeyi, Andrew Nelson, Beth Thompson, Laura Niedernhofer

[DESCRIPTION]
Purpose: Describe the parameters evaluated per FFPE human liver sample received/obtained for the University of Minnesota SenNet TMC. These parameters are used to evaluate whether the tissue can be characterized as "normal" vs "diseased" prior to further protocols.



[STEPS]
1. Prepare two slides with serial sections of the FFPE liver sample -- 1 section per slide.
2. Section A -- Stain with H&E.
Section B -- Stain with trichrome.
4. Schedule evaluation with pathologist (ideally with a strong liver background). 
5. Request assessment and record of the following scores: 
1. Steatosis, macro %
2. Steatosis, score (0, 1, 2, 3) 
3. Ballooning (0, 1, 2) 
4. Inflammation (0, 1, 2, 3)
5. Fibrosis (0, 1a, 1b, 1c, 2, 3, 4)
6. Any comments on preservation/quality of the sample 
7. Other/Additional identifications (e.g. the presence of NASH or other disease states) 
6. Record these values and consider them prior to moving the sample forward for experimentation processing. 
7. Inventory and archive H&E and trichrome stains at room temperature -- Can be scanned if future images are required. 

Inventory and archive FFPE blocks at room temperature. 
3. Mount coverslip.
TITLE: Prepare NGM no peptone plates
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare NGM plates without peptone. </div><div class = "text-block">Peptone is necessary for bacterial growth and thus by not adding peptone, one can control for the number of bacteria present on an NGM plate by seeding a known number of bacteria.</div></div>

[STEPS]
?. 
?. ABC1IngredientsQuantity2NaCl 15g3Double
  distilled H2O4.8L4Agar&nbsp;&nbsp;per 1L bottle17g5Agarper 500ml bottle8.5g
ABC1IngredientsQuantity2NaCl 15g3Double
  distilled H2O4.8L4Agar&nbsp;&nbsp;per 1L bottle17g5Agarper 500ml bottle8.5g
?. A1Measure approx 4.8L&nbsp; double distilled H2O in 5L bell jar with a magnetic flea.2Add NaCl3Stir until all solutes are dissolved.4Dispense approx 972ml&nbsp; NGM media per 1L bottle OR 486ml NGM into a
  500ml bottle.5Dispense 17g of agar and a magnetic flea to each
  1L bottle OR 8.5g of agar and a magnetic flea to each 500ml bottle.6Label, date and autoclave.
A1Measure approx 4.8L&nbsp; double distilled H2O in 5L bell jar with a magnetic flea.2Add NaCl3Stir until all solutes are dissolved.4Dispense approx 972ml&nbsp; NGM media per 1L bottle OR 486ml NGM into a
  500ml bottle.5Dispense 17g of agar and a magnetic flea to each
  1L bottle OR 8.5g of agar and a magnetic flea to each 500ml bottle.6Label, date and autoclave.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Follow the directions to make the best picture ever!</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Sequential Transfection and Transduction Protocol For HUVECs (Lipo2000 version) + HALO tag live microscopy
AUTHORS: Emir  Bora Akmeriç

[DESCRIPTION]
4 Day Protocol for transfecting lipofected plasmids, followed by viral transduction and pre live microscopy treatment. Optimized for blood flow simulation experiments using 0.4 Ibidi uslides with HUVECs.

[STEPS]
SECTION: Pre Transfection
1. Seed 500k cells to a t25 flask a day(to 1.5 days max) prior, the plate on day of transfection should be 50-70% confluent
SECTION: Transfection
3. Prepare the following mixes:

Mix1: 325 uL opti-mem+ 5 uL Lipo2000
Mix2: 325 uL opti-mem + 8 ug Kank1:GFP Plasmid
SECTION: Pre Transfection
2. 2 hours prior to transfection, change media with EGM2 WITHOUT antibiotics!
SECTION: Transfection
4. Incubate for 5 mins at RT
SECTION: Transfection
5. Mix the tubes, incubate for 20 mins at 37C
SECTION: Transfection
6. Add the mix to the semi confluent t25, incubate for 2hours inside the incubator
SECTION: Transfection
7. Exchange the media with 4 mL EGM2(probably without antibiotics), incubate overnight
SECTION: Transduction
8. Warm EGM2 media with antibiotics
SECTION: Transduction
10. Add 2.66 mL EGM2 with antibiotics 
SECTION: Transduction
11. Bring the flask to S2 in a styrofoam box, suit up
SECTION: Transduction
12. Thaw a F1 VEC-Halo AAV, note which tube you are thawing (ideally only thaw a tube 5-6 times)
SECTION: Transduction
13. Add 53 uL of the virus
SECTION: Transduction
14. Incubate overnight
SECTION: Seeding
15. Warm up 2% gelatin in water bath. 
SECTION: Transduction
9. Check cells, ideally with an epifluorescence microscope to see if GFP transfection worked
SECTION: Seeding
19. Wash cells 3x with 4mL of EGM2 and add 4mL media again
SECTION: Seeding
18. Bring a 50 mL falcon of EGM2 without antibiotics to S2, prewarmed.
SECTION: Seeding
16. Dilute 1:10 to a total volume of 300 uL
SECTION: Seeding
17. Add 120 uL of the diluted mix to 2 IBIDI ibitreat luer 0.4 flow slides(uslides), incubate 1-2 hours at 37C
SECTION: Seeding
20. Harvest transduced/transfected cells and bring to a concentration of 2.25 million/mL
SECTION: Seeding
21. Add 100 uL of cell suspension, work fast and avoid bubbles
SECTION: Seeding
22. Incubate for 30 minutes

SECTION: Seeding
23. Add 120 uL of EGM2+antibiotic to the reservoirs, incubate overnight
SECTION: Pre Live Microscopy Treatment
24. Prewarm CO2 independent media with GA antibiotic and thaw a 200 uM Janelia Fluor 549 HaloTAG
SECTION: Pre Live Microscopy Treatment
25. Dilute halotag 1:200 with warm media, incubate for 5 mins. Then dilute 1:5 again.
SECTION: Pre Live Microscopy Treatment
26. Add 220 uL of media per slide. Incubate for 45 mins
SECTION: Pre Live Microscopy Treatment
28. Gently remove media and carefully add an equal volume. Transfer slides to the imaging facility
SECTION: Pre Live Microscopy Treatment
27. Meanwhile, put a styrofoam box in the incubator for moving.
TITLE: A systematic review protocol for Biometrics in healthcare: strategies for improving safety and privacy of patients’ records in sub-Saharan Countriestitled protocol
AUTHORS: Arnold M Hamapa, Joseph Mumba Zulu, oswellkhondowe, Choolwe Jacobs, Mercy Wamunyima Monde, Adam Silumbwe, lydiahangulu, oswellkhondowe

[DESCRIPTION]


Background: The healthcare system is increasingly adopting biometric services to deliver safe, efficient, and cost-effective care. However, security concerns arise when users forget their passwords, share them, jot them down, or store them in close proximity to their computers. Further, some passwords are easily guessable, which allows unauthorized access to entire systems. The aim of this systematic review is to appraise evidence on biometric identification strategies for improving safety and privacy of patient’s records in sub-Saharan African Countries


Methods: A systematic review will be conducted on studies that report on medical record security improvement techniques in African countries. Using relevant search terms, a systematic search for suitable peer-reviewed literature will be done in the following electronic bibliographic databases: PubMed, Theses Global and African Journals Online (AJOL), Cochrane Database of Systematic Reviews, and Google Scholar. An electronic search will be carried out for papers describing biometrics in healthcare: improved patient safety and privacy in African countries.  The search strategy will encompass biometric terms such as fingerprint, facial, or iris recognition for authentication, along with keywords related to patient safety, Africa, healthcare, and patient privacy.


Discussion: This systematic review will provide a thorough assessment of the existing literature, evidence synthesis, identification of best practices, strategies for overcoming barriers and challenges, consideration of ethical and legal issues, cost-effectiveness assessment, identification of research gaps, and serving as educational resources. The findings will contribute to understanding how biometric play a significant role not only in ensuring the privacy and security of healthcare in sub-Saharan Africa. This review will provide guidance and support evidence-based decision-making in a region with specific healthcare opportunities and challenges by synthesizing current knowledge. A systematic review will direct future efforts and where resources should be allocated in the region by identifying areas that need more research. Overall, the findings of this systematic review




[STEPS]
TITLE: CNV Pilot - Subject Candidate Contact Protocol
AUTHORS: Brian Britt, Rose Mary Xavier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the manner in which candidate subjects for the CNV pilot study will be contacted for recruitment.</div></div>

[STEPS]
?. [Contact protocol]
Make a first phone call to the candidate using the IRB-approved phone script
?. [Contact protocol]
Send an e-mail and physical mailer to the candidate using the IRB-approved e-mail template and the IRB-approved mail template
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>1. Extract and purify genomic DNA.</p>
<p> </p>
<p>2. Quantitate the gDNA in samples by spectophotometry.</p>

[GUIDELINES]
<p>1 Keep all reagents protected from light until you are ready to use them. Excessive exposure to light may affect the fluorescent probes.</p>
<p> </p>
<p>2 Minimize freeze-thaw cycles.</p>
<p> </p>
<p>3 Prior to use:</p>
<p>– Mix the TaqMan Genotyping Master Mix thoroughly by swirling the bottle.</p>
<p>– Thaw any frozen TaqMan reagents by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly.</p>
<p>– Resuspend the TaqMan reagents by vortexing, then centrifuge the tube briefly.</p>
<p>– Thaw any frozen genomic DNA samples by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Creating Bacterial Glycerol Stocks for Long-term Storage
AUTHORS: Bonnie Evans

[DESCRIPTION]
Bacterial glycerol stocks are important for long-term storage. Bacteria on LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.

[BEFORE_START]
Make the 50% glycerol solution by diluting 100% glycerol in dH20 and autoclaving.

[STEPS]
SECTION: Steps
1. Grow an overnight culture of the selected strain
 

SECTION: Steps
2. Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryotube and gently mix
SECTION: Steps
4. Freeze the glycerol stock tube at -80°C.

SECTION: Steps
5. To recover bacteria from your glycerol stock, open the tube and use a sterile loop or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw! 
 

SECTION: Steps
3. Shake the glycerol 5-6 times before freezing.
TITLE: Protein expression on the surface of Escherichia coli
AUTHORS: Maja Rennig, Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Andreas Birk Bertelsen, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Display of proteins on the bacterial cell surface has always been an attractive technique for the production of functional cell-anchored proteins, thereby reducing the cost, time and effort related to enzyme purification. Thus, surface display can enable fast and easy screening of protein libraries, e.g. activity variants, or screening of different enzyme substrates, while maintaining a connection between the phenotype and the genotype. Additionally, cells displaying functional enzymes can potentially be used as whole-cell catalysts.</div><div class = "text-block"><span>This protocol describes how to express proteins on the surface of </span><span style = "font-style:italic;">E. coli</span><span> BL21 (DE3) using a L-rhamnose inducible expression system. </span></div></div>

[STEPS]
?. [Pre culture - Day 1]
Pick a fresh colony of your BL21 (DE3) strain with your expression vector, and inoculate it in LB supplemented with relevant antibiotics. Grow the culture at  at 250 RPM shaking . The volume of the overnight culture depends on the volume of the expression culture and should be at least 1/100 of the expression culture
37 °C
?. [Inoculation, Induction and expression - Day 2]
Dilute the overnight culture 1:100 in fresh LB supplemented with relevant antibiotics
?. [Inoculation, Induction and expression - Day 2]
Grow the culture at  with 250 RPM shaking until an OD600 = 0.5 - 0.6
37 °C
?. [Inoculation, Induction and expression - Day 2]
Induce expression by adding L-rhamnose to a final concentration of
?. [Inoculation, Induction and expression - Day 2]
Let the culture grow at   with 250 RPM shaking for up to
30 °C
?. [Assaying surface displayed proteins - Day 3]
Spin the culture down at  and discard the supernatant
Centrifuge: 4000 34, 5 min, 4 10
?. [Assaying surface displayed proteins - Day 3]
Resuspend pellet in the desired assay buffer (e.g.  -  Tris-HCl)
?. [Assaying surface displayed proteins - Day 3]
Run activity assay in liquid or on agar plates.
?. [Alternatively: Shaving-off of surface displayed proteins - Day 3]
Spin the culture down at  and discard the supernatant
Centrifuge: 4000 34, 5 min, 4 10
?. [Alternatively: Shaving-off of surface displayed proteins - Day 3]
Resuspend pellet in  Tris-HCl or /ODU TEV cleavage buffer ( Tris-HCl ,  NaCl2,  EDTA)
100 µl
?. [Alternatively: Shaving-off of surface displayed proteins - Day 3]
Add purified and activated TEV protease
?. [Alternatively: Shaving-off of surface displayed proteins - Day 3]
Incubate at  (~)
0 Room temperature
It is also possible to shorten the incubation time, so that the protein harvest can still be performed on the same day.
?. [Protein harvest - Day 4]
Spin the cells at  and collect the supernatant
Centrifuge: 5000 34, 10 min, 4 10
?. [Protein harvest - Day 4]
Keep the extractions  when working with it and at  for storage
on ice
4 °C
TITLE: Prep for FACS on blood samples
AUTHORS: Grace Burgin, Inbal Benhar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To prepare blood samples for flow </div></div>

[STEPS]
?. Collect blood into EDTA coated tubes
?. Transfer 50ul into clean tube - include 1 unstained***mix from several mice to have 50ul for unstained ctrl tube
?. Add 1ml ACK, incubate at RT for 7 min, centrifuge 5’ 350g
?. Resuspend pellet in FACS buffer + antibody mix
?. Incubate in dark for 15’ on ice
?. Wash (centrifuge) in FACS buffer, 5’ 350g
?. Resuspend in 300ul FACS Buffer + calcein blue