eamag/protocols-with-steps huggingface dataset:
TITLE: W-3 WATER STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes leech storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing field-collected leech samples and nucleic acid samples purified from these soil.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of leech samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.

 
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 

Each collected water sample will be given a unique ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Each water sample filtered membrane will be moved into an individual 60 mm Petri dish labeled with its original sample ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Follow the SOP for water processing (REDI-NET SOP W-2 Water Processing) till the section “Microorganism collection” is completed.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Seal the Petri dish with parafilm and multiple Petri dishes can be stacked together, enclosed with Saran wrap and transferred to a -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
11. Update the freezer inventory so that samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Store water samples for up to 24 hours at 4 °C and up to 7 days at -20 °C before processing the samples.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so that samples can be tracked properly.
TITLE: Keto Blast Gummy Bears Reviews[ Shark Tank] Read Shoking Scams
AUTHORS: Keto  Blast Gummies

[DESCRIPTION]
You have tried to lose weight but the scales won’t move no matter what. What can a girl do? These keto blast gummies are the answer! They are made from all-natural ingredients, and contain only 0.5g sugar per serving.

[STEPS]
TITLE: Suggested field procedures for collecting soil/litter arthropods in a tropical rainforest for long-term monitoring with DNA metabarcoding
AUTHORS: Yves Basset

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Suggested field procedures for collecting soil/litter arthropods in a tropical rainforest for long-term monitoring with DNA metabarcoding.</span></div><div class = "text-block">Standardized field protocols to monitor the soil fauna exist but they are not tailored to surveys in tropical rainforests and not designated to obtain samples that can be later processed with metabarcoding. In general, the soil fauna is extracted by Berlese-Tullgren apparatus, while the litter-dwelling and more active fauna is better surveyed with Winkler extractors. Here, we used Berlese-Tullgren which represent the choice method for Collembola but included in our samples both soil and litter.</div></div>

[STEPS]
TITLE: Protocol name 
AUTHORS: Joanna Sendecka, Joanna Sendecka

[DESCRIPTION]
This is a test protocol created for the purpose of developing guidelines.

[STEPS]
SECTION: Section one
1. Here we start writing the protocol. It will last 10 min

SECTION: Section one
1.1. and here we continue
SECTION: Section one
2. We give examples of amounts e.g. volume 0.5 L or weight 200 g or voltage 3 V

SECTION: Section one
3.
TITLE: Glycine-Sodium Hydroxide Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Glycine-Sodium Hydroxide Buffer]
Mix  and indicated volume of sodium hydroxide solutions.  ABCDEFGH1mL of Sodium hydroxide4.08.816.827.232.038.645.52pH8.69.09.49.810.010.410.6
pH:   to  (a) 0.1 M Glycine; 7.5 g L-1 (M.W.: 75.0 g mol-1)(b) 0.1 M Sodium hydroxide; 4.0 g L-1 (M.W.: 40.0 g mol-1)
[glycine]
ABCDEFGH1mL of Sodium hydroxide4.08.816.827.232.038.645.52pH8.69.09.49.810.010.410.6
?. [Glycine-Sodium Hydroxide Buffer]
Adjust the final volume to  with deionized water.
200 ml
?. [Glycine-Sodium Hydroxide Buffer]
Adjust the final pH using a sensitive pH meter
TITLE: Rotarod Test to assess motor coordination in a mouse parkinsonian model
AUTHORS: natalia.lopezgonzalezdelrey, Zachary Gaertner

[DESCRIPTION]
Protocol for the evaluation of motor coordination in the rotarod assay in parkinsonian mice.

[STEPS]
SECTION: Open Field Test
1. Total duration: 4 days
SECTION: Training phase
3. Duration: 3 days
SECTION: Protocol
2. Training Phase: 3 days

Testing Phase: 3 days
SECTION: Training phase
4. Four trials separated by at least 30 min inter-trial intervals at constant velocity.
SECTION: Test phase
6. Duration: 3 days
SECTION: Test phase
7. Four trials separated by at least 30 min inter-trial intervals in accelerating mode from 4 to 40 rpm in 3000s. 
SECTION: Analysis
9. Latency of fall. The cumulative time (seconds) the subject maintains its balance on the rotarod prior to falling off of the rod. The maximum latency is set at 300 seconds per trial.

The analysis should cover the latency of fall (when applies):
- by day
- by phase
- by group: control vs parkinsonian
- by treatment: control vs l-dopa
SECTION: Test phase
8. Prior to placing the subjects onto the rod, confirm the trip plates are in the active position, the rod is rotating at the start speed of 0 RPM and the reset button has been clicked.
Place the mice onto the rotating rod in sequential order of rod lanes from lane 1 on the far left of the rod to lane 5. Immediately upon all mice being placed onto the rod, press the START button on the instrument panel to start the timers. As the mice fall from the rod, each lane’s trip plate will stop the timer and display the latency in minutes and seconds. Once all mice in a trial have fallen and placed into their cages remove all gross urine and feces with a dry paper towel. The instrument must be thoroughly sanitized with 70% ethanol.
SECTION: Training phase
5. Prior to placing the subjects onto the rod, confirm the trip plates are in the active position, the rod is rotating at the start speed of 4 RPM and the reset button has been clicked.
Place the mice onto the rotating rod in sequential order of rod lanes from lane 1 on the far left of the rod to lane 5. Immediately upon all mice being placed onto the rod, press the START button on the instrument panel to start the timers. As the mice fall from the rod, each lane’s trip plate will stop the timer and display the latency in minutes and seconds. Once all mice in a trial have fallen and placed into their cages remove all gross urine and feces with a dry paper towel. The instrument must be thoroughly sanitized with 70% ethanol.
TITLE: A scoping review of the impact of acquired communication impairments on sexuality, intimacy, and sexual health
AUTHORS: Laura L Wolford, Nicholas Behn, Vanessa Arattia, Emma Power

[DESCRIPTION]
Background
Communication is a crucial part of sexuality. Communication ability has been linked to ability to participate in consent-related discussions, sexual health and safety, sexual self-esteem and self-efficacy, sexual satisfaction, and relationship satisfaction. It therefore makes sense that people with acquired communication impairments would experience changes to their sexual wellbeing as a result of the communication impairment. Additionally, healthcare clinicians may perceive the communication impairment as reducing the client's ability to discuss sexuality and sexual health. This may make sexual wellness even more difficult to access for people with acquired communication impairments. Therefore, this scoping review will be undertaken to better understand the scope of the literature relating acquired communication impairments to sexuality and how these impacts are discussed in the literature. 


Methods 
We will use Arksey and O'Malley (2005) framework for a scoping review. We will search CINAHL, PubMed, MEDLINE, PsycINFO, Web of Science, and Scopus. No date limits will be placed on the search. Additional hand searches of reference lists will be conducted. Data will be extracted relating to the context of the publication, disorder area and communication impairment studied, how communication was discussed in the article, explicitly stated effects of communication impairment on intimacy/sexuality, effects of communication impairment on ability to participate in the study, and any accommodations made for people with communication difficulties. Quantitative data will be explored with descriptive statistics, and qualitative data will be analyzed using a meta-qualitative approach with latent content analysis to interpret meaning and explore themes. 

The full search strategy and methods are available in the PDF attached. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Shows how to create a BLAST db using CyVerse. This example creates a database from E. coli's genome due to size constraints. This protocol is intended to demonstrate the methodology to creat the db but will not have any practicality since the db is only of E. coli's genome. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Plasma FABP4 concentrations were analyzed blinded to their preeclampsia status with BioVendor Human AFABP ELISA (Cat. No: RD191036200R, Biovendor, Modrice, Czech Republic) in our study. The manufacturer reports a normal range of 19.58 ± 16.32 ng/mL (mean ± 2 SD) for 35- to 52-year-old women. The limit of detection of the FABP4 ELISA assay is 0.05 ng/ml. The antibodies used in this ELISA are specific for human AFABP according to the manufacturer.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>These protocols refer to a study having the aim to identify and test at field artificial structures suitable as collectors for common cuttlefish <em>Sepia officinalis</em> eggs in wild condition for restocking purpose.</p>
<p>During the same study, three different protocols were tested and compared. The 'seagrass' collector, the 'longline' collector and the 'trap' collector.</p>
<p>This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).</p>
<p> </p>

[STEPS]
TITLE: Crystallization of Enterovirus D68 3C protease
AUTHORS: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon

[DESCRIPTION]
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. A cause for concern is the currently emerging pathogen Enterovirus D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation. This protocol was used to grow D68 3C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D68 3C.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
14.4 µL35 mg/mL 
2.88 mL 
7.2 µL seeds, dilution 1:1 000 000

SECTION: Crystallization experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 50 286135 mg/mL  to each lens using the SPT mosquito.
Dispense  100 2861 to each lens using the SPT mosquito.
Dispense  25 2861 to each lens using the SPT mosquito.

Drop ratio: 2:4:1 
Final drop volume: 175 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.1 Molarity (M)  Tris pH 7.8 
0.2 Molarity (M)  Ammonium acetate
26% w/v PEG 3350

Stock solutions used:
1 Molarity (M)  Tris adjusted to pH 7.8  with NaOH
1 Molarity (M) Ammonium acetate
50% w/v PEG 3350

 

SECTION: Crystallization experiment
7. 
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito 
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 1 000 000 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.62
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: Total nucleic acids extraction, purification and cDNA synthesis from soil
AUTHORS: Roey Angel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extraction of DNA, and especially RNA from soils, can be challenging due to the presence of organic impurities, which can inhibit downstream enzymatic reactions and the fact that many soil microorganisms are dormant or nearly inactive and hence hard to lyse. The following collection of protocols describe a robust and flexible pipeline for extracting high amounts of pure DNA and RNA from nearly all types of soils. The methods have been successfully tested for mineral and highly organic soils and soils with very low biomass. </div><br/></div>

[STEPS]
TITLE: Meta-analysis of scope harvesting radial artery
AUTHORS: Tzu-yen Huang

[STEPS]
?. Check MeSH terms, set reaserch keywords: radial artery and harvest.
?. Searches in Pubmed, Medline, the Cochrane Library, and EMBASE, to find studies published from January 1974 to July 2019.
?. Articles with comparisons of open and endoscope artery harvesting were included as long as there were adult patients with coronary artery disease undergoing CABG. Systemic-reviews, case series and case reports were excluded. Studies comparing open arery hatvesting and minimal invasive radial artery harvesting were also excluded. Randomized controlled trials (RCTs) and non-randomized observational articles including prospective or retrospective studies were included.
?. Primary outcomes included the wound infection rate, the wound complication rate, harvesting site neurological complications of the forearm during hospitalization, in-hospital mortality, long-term survival (over one year), and the patency rate (duration may be defined differently by research, from in-hospital to 3–5 years).
?. The risk of bias of randomized controlled trials was evaluated by the Cochrane Collaboration risk-of-bias tool—Revised Cochrane risk-of-bias tool for randomized trials (RoB 2).The methodological quality of non-randomized controlled trials (NRCTs) was assessed by the Newcastle–Ottawa Quality Assessment Scale (NOS) .
?. Data synthesis and statistical analysis were conducted using Review Manager
?. Complete study.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Charcoal treatment of seawater for removal of organics.  This is an updated approach to that used by Carlucci &amp; Silbernagiel, 1970.  The approach was used in Paerl et al., 2015 Limnology and Oceanography; DOI: 10.1002/lno.10009

[BEFORE_START]
Obtain: <br />2 designated flasks for charcoal treatment<br />2 stir bars<br />1 filtration rig for charcoal treatment<br />1 GF/F filter (47mm recommended)<br />1 disposable filtration rig with 0.2 μm filter<br />activated charcoal (Sigma C9157 recommended)<br />1 filtration rig with 0.2 μm filter for filter-sterilzing the initial seawater sample

[STEPS]
?. 
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TITLE: Automated immunodetection of proteinase-K-resistant Lewy Pathology in FFPE human colon tissue
AUTHORS: Elizabeth Videlock

[DESCRIPTION]
Automated detection of gut Lewy Pathology using Leica Bond RX processor based on Protocol F using Bond Polymer Refine Detection kit (Leica Biosystems, Cat#: DS9800). Adapted from "Method 5" as described in Beach TG et al., J Parkinsons Dis. 2016.
 


 

Acknowledgments
We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona, for the provision of human colon tissue and data. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinsons Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimers Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimers Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinsons Disease Consortium) and the Michael J. Fox Foundation for Parkinsons Research. 

We wish to acknowledge Yunfeng Li and the Translational Pathology Core Laboratory (TPCL) at UCLA for assistance with tissue processing, IHC staining, and scanning.

[STEPS]
1.  Leica Bond RX routine factory based “Bake and Dewax” protocol.  
2. Enzyme Antigen Retrieval: Incubate in Enzyme 1 solution for 20 min at Room temperature 
   

 

3. Incubate in Peroxide Block for 5 min 
4. Incubate in Bond Wash buffer three times   each.
  
5. Primary antibody: Incubate in 1:10,000
   for 60 min  at Room temperature 
6. Incubate in Bond Wash buffer three times   each.
  
7. Incubate with labeled polymer 10 min 
  
8. Incubate in Bond Wash buffer three times 2 min each.
9. Incubate with polymer for 8 min .
10. Incubate in Bond Wash buffer five times   each.
11. Deionized water wash  .
12. Incubate with Mixed DAB Refine for   followed by Mixed DAB Refine for 10 min.
13. Deionized water wash for three times    each.
14. Incubate with Hematoxylin for 10 min .
15. Incubate in Bond Wash buffer three times   each.
16. Deionized water wash  .
17. Slides are dehydrated in series of alcohols, cleared with   and mounted with
TITLE: Multisite Gateway Calculations: Excel spreadsheet
AUTHORS: Christian  Mosimann

[DESCRIPTION]
Multisite Gateway cloning provides a modular tool to assemble plasmid vectors suitable for numerous applications. The assembly of Multisite Gateway plasmids requires the correct ratios of three entry vectors and the destination backbone to favor correct recombination by Gateway LR Clonase II Plus. While simple, the calculation of recombination mixes is a reoccurring task when using Multisite Gateway that benefits from automation and proper documentation. This protocol provides a simple Excel-based spreadsheet to simplify the involved calculations based on providing individual vector lengths and working stock concentrations. Using this Multisite Gateway calculations spreadsheet supports correct reaction mix assembly, scaling of cloning reactions, cloning documentation, and troubleshooting.

[STEPS]
TITLE: IHC Fluorescent Frozen Sections
AUTHORS: Elizabeth Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures.</div></div>

[STEPS]
?. Day 1: Using the PAP Pen, carefully draw a water barrier circle around the tissue sections on the slide – allow this circle to dry for several seconds or up to approx. one minute
?. Rinse slides with PBS (pH 7.3-7.4): 4 x 5 min each
?. Rinse slides with 0.5% BSA + 0.4% Triton X-100 in PBS):  1 x 10 min
?. Remove slides one at a time and using a clean Kimwipe, carefully wipe around the tissue sections to dry the slide
?. Place the slides into a black, covered slide incubation box/humidity box
?. Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
?. Pour off the blocking buffer
?. Replace with primary antibody solution (antibody of choice diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Incubate tissue with primary antibody overnight in incubation box
?. Day 2: Rinse slides with PBS:  4 x 5 min
?. Rinse slides with 0.5% BSA + 0.4% Triton X-100 + PBS:  1 x 10 min
?. Place the slides into a black, covered slide incubation box/humidity box
?. Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
?. Prepare fluorescent secondary antibody (secondary antibody should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Cover the tissue with the secondary antibody solution and incubate for 2 hrs. at RT in the incubation box. *From this point on, use low light and/or cover tissues.*
?. Rinse slides with PBS:  4 x 5 min
?. Remove excess PBS with a Kimwipe
?. Carefully add a drop of mounting medium to the center of the tissue and apply cover glass
?. Seal cover glass with clear nail polish. For thicker tissue, add a weight before sealing
TITLE: Hornwort sporophyte induction - Bonn
AUTHORS: Eftychis Frangedakis

[STEPS]
?. Grow small thallus fragments for two weeks in petri dishes on Knop or BCD media at pH 5.7 and containing 0.7% (w/v) Gelzan (A in figure).
?. Transfer plants to Magenta pots on Knop medium or BCD medium (B and C in figure)at pH 5.7, containing 0.7% (w/v) Gelzan. Add 2mL of sterile water into the pot using a pipette.
?. Place pots in a Panasonic MLR-352 Versatile Environmental Test Chamber (or similar growth chamber / tissue cluture room)  at 21oC, 12 h of light and 12 h of dark, 1500 lux light intensity.
?. After approximately one month, antheridia start to appear. Add another 1-2 mL of sterile water into the pot using a pipette  (D in figure).
?. After one more month sporophytes emerge (E in figure).
?.