eamag/protocols-with-steps huggingface dataset:
TITLE: Chloroform Phenol Phage genome isolation
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol of the isolation of genomic DNA from phage lambda and phage T7. </div></div>

[STEPS]
?. Use cleaned phage stock from the phage stock protocol. Add  DNase I 10x buffer, DNase I (1 U/µL), and  RNase A (10 mg/mL) for  at  without shaking to remove E. coli DNA and RNA.
50 µl
1 µl
1 µl
37 °C
?. Add of  EDTA (final concentration ) and incubate for  at  to deactivate DNase I and RNase A
20 µl
75 °C
?. Add  Proteinase K (20 mg/mL) and  SDS 10% to digest the phage capsid and incubate overnight at  without shaking.
2 µl
50 µl
56 °C
?. Add equal volume phenol/chloroform/iosamyl alcohol (25:24:1) and mix well (do not vortex, as genome is easily damaged)
?. Centrifuge at room temperature for  at 10000 x g
?. Carefully take aqueous phase
?. Add equal volume chloroform/isoamyl alcohol (24:1) and mix well (do not vortex, as genome is easily damaged)
?. Centrifuge at room temperature for  at 10000 x g
?. Carefully take aqueous phase
?. Add 1/10 volume of Sodium acetate
?. Add 2,5 volume of 100% Ethanol
?. Incubate at  for
-20 °C
?. Centrifuge for at 4668 x G
?. Discard supernatant
?. Rinse pellet with 70 % ethanol
?. Dry pellet. Heating to    increases drying.
50 °C
?. Dissolve pellet in MQ water
TITLE: Immunohistochemistry of liver tissue sections
AUTHORS: Presha  Rajbhandari, Taruna Neelakantan, Brent R. Stockwell

[DESCRIPTION]
This protocol outlines the steps used to perform standard immunohistochemistry for antibody validation in frozen human liver tissue samples, performed at Molecular Pathology Core facility at Columbia University.

[STEPS]
1. Cryosection the frozen liver tissue at 5µm thickness and place it on charged slide

2. Air dry the sections for 3 minutes 180 min 

3. Fix the tissue sections in cold acetone for 15 minutes 15 min . Alternatively, fix with 1% paraformaldehyde at 4C for 15min followed by ice-cold methanol at -20C for 5min.

4. Air dry the sections at room temperature for 2 minutes 2 min  

5. Incubate the slides in 0.3% hydrogen peroxide in PBS for 5 minutes 5 minto block peroxidase activity

6. Wash slides with PBS 3 times, for 5 minutes each 5 min 

7. Block the tissue sections in 10% normal goat serum or 5%Horse serum with 0.1%BSA for 20 minutes 20 min 

8. Incubate the tissue sections with primary antibody diluted in DAKO antibody diluent at room temperature for 1.5-2 hours 90 min 120 min 

9. Wash the slides with PBS 3 times, for 5 minutes each  5 min 

10. Incubate the tissue sections with biotinylated secondary antibody diluted in PBS at room temperature for 30-45 minutes 30 min 45 min 

11. Wash the slides with PBS 3 times, for 5 minutes each  5 min 
12. Incubate the tissue sections with ABC (Avidin-Biotin complex) peroxidase solution at room temperature for 30 minutes  30 min 

13. Wash the slides with PBS 3 times, 5 minutes each  5 min 

14. Incubate the tissue sections with DAB (3,3’-diaminobenzidine) peroxidase substrate solution until desired color intensity is reached and immerse slides in distilled water
15. Counterstain with hematoxylin and rinse with distilled water
16. Dehydrate the sections using 95% ethanol followed by 100% ethanol
17. Clear with xylene and mount coverslip using mounting medium
TITLE: QUESTIONNAIRE ABOUT ECO METHOD IMPLEMENTATION IN THE UNIVERSITY
AUTHORS: Juan Jesus Torres-Gordillo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The purpose of this questionnaire is to know the impact of the implementation of the ECO method in the University. It is part of an innovation project, entitled </span><span style = "font-style:italic;">ECO in Higher Education. Teaching inspired by the environment</span><span>, funded by the University of Seville's 3rd Teaching Plan. It is intended for teachers who have been part of the innovation project. The data collected will be analysed to be used in the final report, as well as in some conference communications or journal articles.</span></div><div class = "text-block">The questionnaire consists of 29 questions, divided into three blocks: 12 initial questions on demographic data, 9 closed-ended questions with four options and 8 open-ended questions. For open-ended questions, we ask that you answer in detail and as explicitly as possible.</div><div class = "text-block">Each teacher is requested to complete one questionnaire per course in which they have implemented the ECO method. Similarly, if there have been two teachers in a shared course, each must fulfill, from their experience, a separate questionnaire.</div><div class = "text-block">Thank you very much for your collaboration.</div><div class = "text-block">Prof. Juan-Jesús Torres-Gordillo</div><div class = "text-block">University of Seville (Spain)</div></div>

[STEPS]
TITLE: Acetone precipitation of proteins
AUTHORS: iGEM Dusseldorf

[STEPS]
?. Add 4 times the volume -20°C acetone to the extract and vortexed
?. Incubate sample at -20°C for 2 hours
?. Centrifuge at 14000 x g for 4 min at 4°C and discard the supernatant
?. Wash pellet twice with a 4:1 acetone/water mixture at -20°C until the pellet is well broken up
?. Centrifuge at 14000 x g at 4°C for 10 min between each washing step  (2x)
?. After final centrifugation, incubate solutaion at 40°C until approximately 80% of the liquid evaporated
?. Resuspend pellet in remaining liquid. Store at -80°C for long term use or -20°C for short term use
TITLE: Experiences of oldest-old caregivers whose partner is approaching end-of-life: a mixed-method systematic review and narrative synthesis
AUTHORS: Tessa Morgan, Aamena Bharmal, Robbie Duschinsky, Stephen Barclay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background and Objectives:</span><span> Ageing populations have rapidly increased the number of people requiring end-of-life care across the globe. Governments have responded by promoting end-of-life in the community. Partly as a consequence, older spouses are frequently providing for their partner’s end-of-life care at home, despite potentially facing their own health issues. While there is an emerging literature on young-old carers, less is known about spouse carers over 75 who are likely to face specific challenges associated with their advanced age and relationship status. The aim of this review, therefore, is to identify and synthesise the literature concerning the experiences of carers aged 75 and over whose partner is approaching end-of-life. We conducted a mixed-method systematic review and narrative synthesis of the empirical literature published between 1985 and May 2019, identified from six databases: Medline, PsychINFO, Cumulative Index to Nursing and Allied Health Literature, Embase, Sociological Abstracts and Social Service Abstracts. Gough’s Weight of Evidence and Morgan’s Feminist Quality Appraisal tool used to determine the quality of papers. From the initial 7760 titles, 10 qualitative studies and 9 quantitative studies were included. We identified three themes: 1) “Embodied impact of care” whereby caring was found to negatively impact carers physical and psychological health, with adverse effects continuing into bereavement; 2) “Caregiving spouse’s conceptualisation of their role” in which carers’ navigated their self and marriage identities in relation to their partner’s condition and expectations about gender and place; 3) “Learning to care” which involved learning new skills and ways of coping to remain able to provide care. We have identified a recent up-surge in published papers about very old spousal carers, which comprise a small, medium-quality evidence base. This review outlines a range of potential lines of inquiry for future research including further clarification of the impact of caregiving on the likelihood of mortality, the incidence of men and women providing end-of-life care amongst this age group, and the role of anticipatory grief in shaping their perceptions of their relationship and their own longevity. </span></div></div>

[STEPS]
TITLE: Study of prescription-indication of antivirals for herpesviruses in a Colombian population. A cross-sectional study
AUTHORS: Jorge Machado Alba

[DESCRIPTION]
Introduction:
Herpesviruses are among the most common pathogens worldwide.
 
Objective: To
determine the prescription patterns and indications for the use of antivirals
for herpesviruses in a group of patients from Colombia.
 
Methods: This was a
cross-sectional study on the use of antivirals for treating outpatients with
herpesviruses between November 2023 and January 2024 in a Colombian population
database. The approved and unapproved indications were determined according to
the records of the Food and Drug Administration (FDA) and the National
Institute of Food and Drug Surveillance (INVIMA) of Colombia. Descriptive
analysis was carried out.
 
Results: In total, 17,275
patients were identified, with an average age of 47.5 ± 21.2 years, and 59.1%
were women. The most frequently prescribed antiviral was acyclovir, and the
most frequently used antiviral agent was herpes zoster (47.6%). In total, 9.8%
of the prescriptions were used for unapproved indications, with vaginitis/vulvitis/vulvovaginitis
(3.2%) standing out. In total, 55.2% were treated only with oral dosage forms,
24.6% with oral and topical dosage forms, and 20.2% with only topical dosage
forms. The concomitant prescription of systemic (17.0%) and topical (8.5%)
antimicrobial agents was common.
 
Conclusions:
Acyclovir was the most commonly prescribed antiviral for the management of herpes
zoster. Adherence to clinical practice guidelines was not adequate in some
cases, indicating unapproved uses and uses in entities where efficacy is very
doubtful or limited, especially with topical pharmaceutical forms. The
concomitant prescription of other antimicrobials was common.

[STEPS]
1.
TITLE: Protocol for breeding Hymenochirus boettgeri in captivity
AUTHORS: Tamilie Carvalho, Timothy Y. James

[DESCRIPTION]
Hymenochirus boettgeri is a sexually dimorphic species, with females ty pically exhibiting a larger oval-shaped body (up to 35 mm in snout-vent length (SVL)), while males possess a large obiculated tympanum and a lateral post-axillary subdermal gland that increases in size and vascularity during sexual activity (Figure 1).  


[GUIDELINES]
Conclusion:

This protocol outlines the steps for breeding H. boettgeri in captivity. Careful selection of breeding pairs, proper feeding, and attentive observation are essential for successful breeding and egg collection. Regular water changes help maintain water quality during the breeding period.

[STEPS]
SECTION: Sexual selection
1. Select male H. boettgeri specimens with heavily vascularized glands and the largest females for breeding.
 

SECTION: Sexual selection
2. Keep males and females in separate 38 L glass tanks by sex at 25 °C with conditioned water as described in the “Housing and Care for Hymenochirus boettgeri” protocol and 13:11 light-dark cycle.
SECTION: Feeding
3. Feed the frogs ad libitum twice per day for ten consecutive days, providing newly hatched brine shrimp as the first meal of the day, and offer either frozen gamma-irradiated bloodworms, live blackworms, or small-granule fish food as the second meal of the day, changing the combination daily if possible.
SECTION: Pair selection
4. After 10 days of enhanced feeding, choose breeding pairs consisting of one male and one female each.
SECTION: Mating tanks
5. Using an aquarium net, place each breeding pair in separate 8 L plastic tanks at 21 °C, with a minimum water volume of 2 L per individual.
 

SECTION: Mating tanks
6. Allow the breeding pairs to remain together for one to four days, during which mating behavior is expected.
SECTION: Mating tanks
7. Observe the frogs closely during this period. If the pair does not reproduce, consider replacing one of them with another animal to create a new pair combination.
SECTION: Egg Collection and maintenance
8. After mating, collect the eggs as they are laid using a sterile siphon or spoon and transfer them to net breeder tanks as described in “Housing and Care for Hymenochirus boettgeri” protocol.
SECTION: Egg Collection and maintenance
9. Check the eggs daily and remove any that contain fungi growing or that haven't hatched after 4 days of being laid.
SECTION: Water safety
10. During the breeding period, feed the animals and, if necessary, perform a water change as described in the “Housing and Care for Hymenochirus boettgeri” protocol.
TITLE: 2-step PCR mixture and conditions (Barcoded-head primers for seqs pooling)
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
1. Wear glove, clean up the working bench w. 1% bleach
SECTION: For 1&#x27; PCR head-primers
2. Prepare 1' PCR master mixutre for head-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for head-primers for each reaction
 

 

SECTION: For 1&#x27; PCR head-primers
3. Mix the 1' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 1&#x27; PCR head-primers
4. Transfer  15 µL 1' PCR master mixutre in 8-strip PCR tubes.

SECTION: For 1&#x27; PCR head-primers
5. Add 0.6 µLDNA template in 8-strip PCR tubes, resulting in a 15.6 µL reaction mixture for 1' PCR.

Negative control contains only 15 µL master mixture but not DNA template

SECTION: For 1&#x27; PCR head-primers
6. Mix the reaction mixture gently by tapping the tubes. Quick spin the tubes.
SECTION: For 1&#x27; PCR head-primers
7. Carry out PCR using the following condition:  

1' PCR condition for head-primers
 

SECTION: For 1&#x27; PCR head-primers
7.1. 1' hear-primers used in Huang lab
 

SECTION: For 1&#x27; PCR head-primers
8. Carry out electrophoresis for inspection of DNA products

   

SECTION: For 1&#x27; PCR head-primers
9. Markdown wells and upload the pictures to the Lab Google drive

  

SECTION: For 2&#x27; PCR barcoded-head primers
10. Prepare 2' PCR master mixutre for barcoded-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for barcoded-primers for each reaction (NO PRIMERs at this point!!)
 

 

SECTION: For 2&#x27; PCR barcoded-head primers
11. Mix the 2' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 2&#x27; PCR barcoded-head primers
12. Transfer 21.5 µL of the 2' PCR master mixture to 8-strip PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
13. Add 2.5 µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
14. Add 1 µL of 1' PCR product as template, resulting in 25 µL reaction mixture for 2' PCR.

Negative control contains only 24 µL master mixture and premixed barcoded-head primers but not DNA template
SECTION: For 2&#x27; PCR barcoded-head primers
15. Mix gently by tapping the tubes. Quick spin the tubes.
SECTION: For 2&#x27; PCR barcoded-head primers
16. Carry out 2' PCR using the following condition:  

2' PCR condition for barcoded-head primers
 

SECTION: For 2&#x27; PCR barcoded-head primers
16.1. 2' barcoded-head primers used in Huang lab
 

SECTION: For 2&#x27; PCR barcoded-head primers
17. Carry out electrophoresis for inspection of DNA products

  

SECTION: For 2&#x27; PCR barcoded-head primers
18. Markdown wells and upload the pictures to the Lab Google drive
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The RCC Team, Station Biologique de Roscoff, CNRS/Université P. &amp; M. Curie, Place G. Teissier, 29680 Roscoff, France</p>

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

[DESCRIPTION]
<p>This protocol describes the RT-PCR from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<p> </p>
<p>Please see the manuscript for the full method details.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: pasefRiQ_V1
AUTHORS: lcmsmethods  , Yuting Yuan, Namandje' N Bumpus

[DESCRIPTION]
We have developed optimized methods for reporter ion quantification on a TIMSTOF Flex (TIMS B configuration) by iterative analysis of the TMT triple knock out yeast standard and an in-house developed two-proteome standard.  For the greatest reduction in background coisolation interference in the TMT6 and TMT10/11 plex standards, 10 ion mobility ramps from 0.8 to 1.3 1/k0 provides the greatest reduction in interference on 60 minute LC gradients when using a 25cm IonOpticks aurora column. Reducing the number of ramps to 5 leads to an overall increase in peptide identifications with a loss in unique protein identifications. It is worth noting that the distribution of 1/k0 of peptides labeled with the TMT6/10/11plex and TMTPro reagents have somewhat different overall distibutions. For the maximum intrascan linear dynamic range. For 30 minute pasefRiQ experiments, 10 ramps at a 2.2 second cycle time has a detrimental effect on the overall identification of PSMs, peptides and proteins. 
For the highest possible coverage of each protein identified with the TMTPro reagents on 30 minute gradients we recommend 5 ramps using a 1/k0 range of 0.7 to 1.4. 
Typical results for pasefRiQ using a two proteome standard as described is 3,600 quantified proteins in 60 minutes with a 240ng total load on column. 
At 24ng load on column, this same method with no alterations will return approximately 2,100 quantified proteins. Increasing the multiplier voltage with the high sensitivity model will result in an approximately 15% more peptides and proteins identified at this load. 

[STEPS]
TITLE: Agarose gel for RNA and DNA
AUTHORS: kaylee Beine

[DESCRIPTION]
General method for running agarose gels for RNA and DNA samples

[STEPS]
SECTION: Preparing the agarose gel and plate
1. 1 g  agarose in 100 mL of   

SECTION: Preparing the agarose gel and plate
2. Microwave solution in a flask for 3 min until the agarose is dissolved. Microwave for 1 min thereafter 30 s 

SECTION: Preparing the agarose gel and plate
3. Allow to cool before adding 1 µL   in the gel medium before completely cool. Then pour into the gel mold

SECTION: Preparing the agarose gel and plate
3.1. Make sure that your plate is balanced and the teeth are in. Decide if you need a long ladder or a short weather.
SECTION: Preparing the chamber
4. Add 5 µL   in the gel medium within the chromatography chamber

SECTION: Preparing RNA samples
5. 250-300 ng C1.V1 = C2.V2

V1 = (10)(50)/RNA conc. (from nanodrop)

Add the V1 sample of RNA to 1:1 ratio of   to each sample

SECTION: Preparing RNA samples
6. Heat up the ladder   and RNA samples to 70 °C for 5 min for the gel

SECTION: Preparing DNA samples
7.   is already in the samples for the PCR; then use the   

SECTION: Setting up the sample gels
8. Add 5 µL of ladder and samples to gel wells

SECTION: Setting up the sample gels
9. For RNA: 80V for 60 min 
For DNA: 80V for 40 min 

SECTION: Setting up the sample gels
10. *Note: can check gel after 10 min to see if there is product showing on the gel
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Wet-mount-Method-for-Enumeration-of-Aquatic-Viruse-c8pzvm" target="_blank">Wet-mount Method for Enumeration of Aquatic Viruses.</a>&nbsp;

[GUIDELINES]
This solution is needed only if analyzing unconcentrated samples.

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Final concentration <br />2 &micro;l 10x Buffer 1x <br />2 &micro;l ATP (100 mM) 10 mM <br />2&micro;l GTP (100 mM) 10 mM <br />2 &micro;l CTP (100 mM) 10 mM <br />2 &micro;l UTP (100 mM) 10 mM <br />8 &micro;l DNA template (85 ng/&micro;l) &larr;PCR PDTS 25 ng/&micro;l <br />2 &micro;l T7 RNA polymerase mix <br />20 &micro;l total

[STEPS]
TITLE: Mitophagy induction using Difereprone
AUTHORS: Louise Uoselis

[DESCRIPTION]
Mitophagy induction in HeLa cells using Difereprone (DFP).

[STEPS]
SECTION: Day 1
1. Seed cells, aiming for a confluency of 80-90% at the time of treatment the next day. 
SECTION: Day 2
2. Feed cells for 60 min in an appropriate volume of standard growth media. 

SECTION: Day 2
3. During the feed, warm up the difereprone (DFP) stock aliquot in a 37 °C waterbath. Centrifuge the stock tube after thawing to ensure the DFP has returned to solution. 

SECTION: Day 2
4. To start the treatment, replace the media in each well with standard growth media that contains 1 millimolar (mM) DFP. 

SECTION: Day 2
5. Harvest the samples after the desired treatment times.
TITLE: HuBMAP TMC-Florida/Zurich CODEX  Modality Overview
AUTHORS: Marda Jorgensen, Jerelyn Nick, Franchesca Farris, Jesus Penaloza

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is an overview of all of  the protocols currently in use for the  CODEX modality at HubMAP Tissue Mapping Center,TMC-Florida/Zurich.  It includes protocols.io links to each of the individual protocols that make up this project workflow, from processing tissue and  imaging the samples, to uploading the final data to the HuBMAP HIVE.</div></div>

[STEPS]
?. Process donor organs into samples for analysis. Lymph Node: dx.doi.org/10.17504/protocols.io.bbgnijveThymus: dx.doi.org/10.17504/protocols.io.bbgmiju6Spleen: dx.doi.org/10.17504/protocols.io.bc3kiykw
?. Register organ donor, organs received and common coordinate region information in the HuBMAP UUID generator at https://uuid.hubmapconsortium.org.
?. Prepare paraffin blocks from tissue samples. Cassette Processing: dx.doi.org/10.17504/protocols.io.8nzhvf6Paraffin Embedding: dx.doi.org/10.17504/protocols.io.bam9ic96
?. Prepare H&E sections for histopathological assessment.Sectioning: dx.doi.org/10.17504/protocols.io.bahdib26H&E Staining: dx.doi.org/10.17504/protocols.io.beamjac6
?. Validate antibodies in all target tissue types by IHC using DAB Staining:https://dx.doi.org/10.17504/protocols.io.bdiei4be
?. Coat coverslips with Poly-L-Lysine.Coverslip Preparation: dx.doi.org/10.17504/protocols.io.baxyifpw
?. Cut and mount sections for CODEX analysis. Sectioning: dx.doi.org/10.17504/protocols.io.basdiea6
?. Stain coverslips with Akoya invertoried antibodies. Staining: dx.doi.org/10.17504/protocols.io.bbsdina6
?. Make CODEX bar coded reporter trays. Preparation: dx.doi.org/10.17504/protocols.io.bc2riyd6
?. Perform multiplex data acquisition using CODEX robot and Keyence microscope. Acquisition: dx.doi.org/10.17504/protocols.io.bdifi4bn
?. Transfer images to analysis computer and perform CODEX processing.
?. ​Register completed CODEX section information in the HuBMAP UUID generator at https://uuid.hubmapconsortium.org.
?. Prepare and upload data to the HuBMAP HIVE  Globus using hipergator.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We developed a mini-barcode based on the 12S rRNA gene to identify predator from scats in Tasmania. </p>
<p>To test the sensitivity of our primers to detect low template DNA samples, we set up serial dilutions of six DNA extracts originating from museum samples, representing each of the six mammal predators that might be detected in Tasmania (Tasmanian devil, eastern quoll, spotted tail quoll, cat, dog and fox).</p>

[STEPS]
?. 
?. 
?. 
?.