eamag/protocols-with-steps huggingface dataset:
TITLE: Building and Using the Illuminated Orbital Shaker
AUTHORS: Jakub Nedbal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The building of a bottom-illuminated orbital shaker designed for the cultivation of microalgae suspensions is described in this protocol collection. The instrument agitates and illuminates microalgae suspensions grown inside flasks. It was optimized for low production cost, simplicity, low power consumption, design flexibility, consistent, and controllable growth light intensity.</div><div class = "text-block"><span>The instrument has been used to grow fresh-water microalgae </span><span style = "font-style:italic;">Desmodesmus quadricauda</span><span> and </span><span style = "font-style:italic;">Chlorella vulgaris</span><span>. The cultivation protocols are included in this collection.</span></div></div>

[STEPS]
TITLE: Phosphate Buffered Saline (PBS)
AUTHORS: Andreas Sagen

[DESCRIPTION]
Phosphate-buffered saline (PBS) is a buffer solution (pH ~7.4) commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and optionally, potassium chloride and potassium dihydrogen phosphate. The buffer helps to maintain a constant pH. PBS has many uses because it is isotonic and non-toxic to most cells[1].

[BEFORE_START]
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated

[GUIDELINES]
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.

[STEPS]
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1. All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.1. Fill the bottle with 50 mL double-distilled water
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.2. Measure 8000 mg Sodium chloride, 200 mg Potassium chloride, 144 mg Disodium phosphate and 245 mg Monopotassium phosphate

Powdered compounds:
 
 
 
 
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.4. Adjust pH while mixing to pH 7.4 using concentrated sodium hydroxide
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.5. Add distilled water to a total of 100 mL
SECTION: 100 mL 10x PBS w/o Ca2++, Mg2++
1.6. Autoclave liquid at 121 °C for 15 min
TITLE: F-3 FECES STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes feces storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing feces samples and nucleic acid samples purified from these samples.

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 fecal 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. 
 
Precool 96-well microfuge tube racks on ice.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Using preprinted adhesive labels or permanent markers, label 1.5 ml microfuge tubes with unique sample ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Once the rack is full or all feces samples have been completed, label the rack with a unique rack ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so samples can be tracked properly.
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, make sure to keep samples in the exact same locations corresponding to the rack where the original blood were stored.
 

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 samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Transfer fecal sample into the corresponding pre-labeled 1.5 mL microfuge tubes and put it onto the microfuge tube rack on ice sequentially.
TITLE: Marmoset NHP Free Floating Anti-GFP Antibody Stain After HCR FISH
AUTHORS: Guoping Feng Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for staining NHP tissue for anti-GFP antibody after staining for markers of intrest with HCR FISH.</div></div>

[STEPS]
?. [Primary Antibody]
Permeabilized marmoset brain sections (, fixed previously in  PFA) in  Triton X-100 in PBS for  .
?. [Primary Antibody]
Block sections with  NS (natural serum depending on Secondary Antibody) and PBT [ Triton X-100, 1X PBS] for .
?. [Primary Antibody]
Incubate primary antibody (1:500 whole mount - ) with  NS and PBT-Azide [ NaN3, 1X PBT] at  for .
4 °C
?. [Secondary Antibody]
Wash sections in 1X PBS for  6X.
?. [Secondary Antibody]
Incubate with secondary antibody (1:500 whole mount - ) in PBT at  for .
4 °C
?. [Secondary Antibody]
Wash sections in wash buffer for  6X.
?. [Secondary Antibody]
Stain for DAPI (1:10000 in PBS - ).
?. [Secondary Antibody]
Wash sections in 1X PBS for  2X.
TITLE: Opentrons Pipeline: DNA Extraction with the Mag-Bind Blood &amp; Tissue DNA HDQ 96 Kit Tissue Protocol
AUTHORS: Thalía Silvestre

[DESCRIPTION]
This protocol is an automated pipeline to extract a full 96-well plate of DNA from tissue lysates.
This protocol was developed and optimized for the following:
Platform: Opentrons OT-2 automated pipetting robot
Kit: Mag-Bind‱ Blood & Tissue DNA HDQ Prefilled 96 Kit 
Recommended number of samples: 96 
Two ellutions: Firts ellution( 60μL ) , Second ellution ( 30μL)


[STEPS]
SECTION: List of materials to start to use OT-2
2. It is necessary to have this equipment to use this pipeline.
Opentrons Equipment List:
 


SECTION: List of materials to start to use OT-2
3. Tips & Labware:

 



SECTION: Samples Lysis
1. Preparate 96 samples lysis following the Mag-Bind‱ Blood & Tissue DNA HDQ 96 Kit Tissue Protocol:


SECTION: Star OT-2 run
4. The pipeline start on the step number 7 of the Mag-Bind‱ Blood & Tissue DNA HDQ 96 Kit Tissue Protocol
Load the pipeline on the Opentrons app:   
The VWR 96-Well Deep Well Plate (1000 μL) labware can be  download from: dx.doi.org/10.17504/protocols.io.dm6gp39njvzp/v1



SECTION: Samples Lysis
1.1. Mince up to 10 mg tissue and transfer to a 1.5 mL tubes (one for each sample).
(Cut each 10 mg sample with sterilized cutters as small as possible)

SECTION: Samples Lysis
1.2. Add 250 μL TL Buffer to each sample.
SECTION: Samples Lysis
1.3. Add 20 μL Proteinase K Solution to each sample. Vortex to mix.
SECTION: Samples Lysis
1.4. Incubate at 55°C  overnight in a shaking water bath.
SECTION: Samples Lysis
1.5. Centrifuge at maximum speed (≥4,000 x g) for 5 minutes to pellet undigested tissue
debris and transfer 200 µL of the supernatant to the VWR 96-Well Deep Well Plate (1000 μL) without
disturbing the undigested pellet

SECTION: Star OT-2 run
4.1. Order of the reagents on the Two Nest 12 Well Reservoirs (15 ml)

Slot 1:
 
Slot 4:
TITLE: Modified Rapid Phytolith Methods
AUTHORS: Kali R Wade

[DESCRIPTION]
Processing procedures here are modified from those outlined in Katz et al 2010. Samples can be processed in this way and, following completion, rinsed with deionized water and dried to a stable state for storage and to make extra slides for morphological IDs. 

[BEFORE_START]
Follow the "fail-safe" practices outlined here, in addition to any other health and safety paperwork or contamination recommendations from your organization. 

[STEPS]
SECTION: Fail-Safe Practices
1. Wiping down all equipment, surfaces, and tools used with soap and water followed by acetone (beginning and end of every day of the project).
SECTION: Fail-Safe Practices
2. Checking and adjusting the density of sodium polytungstate (SPT) to conform to the protocol being used (normally between 2.3 and 2.5 g/ml).
SECTION: Fail-Safe Practices
3. Plating one slide of each hydrocloric acid (HCl) SPT, and the mounting agent (likely Cargille Immersion Oil, Type B) and viewing them under microscopy to ensure no contamination.
SECTION: Fail-Safe Practices
4. Calibrating analytical balances.  
18. Inventory samples.
SECTION: Sample Preparation
6. Label 1.5 ml centrifuge tubes (2 per sample being processed) with sample ID.
SECTION: Sample Preparation
8. Weigh 30-80 mg of sediment into 1.5 ml centrifuge tube. Ensure balance is tared following the weight of the tube, prior to weighing sediment, with balances' scale and lab doors closed.
SECTION: Sample Preparation
5. If necessary, place samples in desiccator overnight, set to 30°C.
SECTION: Sample Preparation
7. Sieve samples through 0.5 mm mesh, rinsing mesh between each sample with soap and water, acetone, then deionized water. 
SECTION: Organic and Carbonate Removal
9. Add 50μl of 6N HCl, waiting for bubbling to cease (give the samples a few minutes). Shake tube to ensure chemical gets through all of the sample
SECTION: Organic and Carbonate Removal
10. Add 450μl of 2.5 g/ml SPT
SECTION: Breaking up Soil Aggregates &amp; Creating Pellet
11. Vortex for 3 sec, sonicate for 10 min, vortex 3 sec
SECTION: Breaking up Soil Aggregates &amp; Creating Pellet
12. Centrifuge for 10 min at 6rpm
SECTION: Plating Slides
13. Remove supernatant to clean 1.5ml test tube. Be mindful of proper labels. 
SECTION: Plating Slides
14. Vortex supernatant for 3sec
SECTION: Plating Slides
15. Pipette 50μl of supernatant onto a labelled microscope slide and cover with a coverslip. Seal with nail polish. Slides last between 6-12 hours before crystallization
TITLE: Scott&#39;s Tap Water Substitute
AUTHORS: joshdm

[DESCRIPTION]
Used as a bluing agent in H&E staining

[STEPS]
SECTION: Measure and mix salts
1. weigh 20 g of   

SECTION: Measure and mix salts
2. weigh 2 g of    

SECTION: Measure and mix salts
3. Mix in ddWater until completely dissolved
store at room temperature
pH 8.3 to pH 8.7
TITLE: Workflow for bulk RNAseq of human fallopian tube and uterine endomyometrium 
AUTHORS: Scott Lindsay-Hewett, Valentina Stanley, Mana Parast, Louise Laurent

[DESCRIPTION]
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate bulk RNAseq data from human fallopian tube and uterine endomyometrium.

[STEPS]
SECTION: Tissue preparation
1. As soon as possible after sterilization (salpingectomy or tubal ligation), prepare fallopian tube tissue according to the following protocol:

Human Pregnant Fallopian Tube Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC

At the time of C-section, prepare uterine endomyometrium tissue according to the following protocol:

Human Pregnant Uterine Myometrium Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC

For this protocol, use tissue that has been collected in RNAlater.
SECTION: Total RNA isolation
2. Isolate total RNA using a bead beater to disrupt the tissue, followed by organic extraction and ethanol precipitation. Use the following protocol, which was originally written for placenta:

Total RNA extraction from frozen placenta tissue

After passing quality control, proceed to library construction.

 

SECTION: Library construction
3. Construct libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR), according to the following protocol, which was originally written for placenta:

Library construction for human placenta bulk RNAseq

 

After passing quality control, proceed to sequencing.
SECTION: Sequencing
4. For HuBMAP bulk RNAseq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration. Reads were aligned using STAR, and transcript abundances were quantified using RSEM.
TITLE: Postoperative Rehabilitation of Total Knee Arthroplasty
AUTHORS: Luisa Mululo, Fabricio Loures, Ana Carla Gonzaga de Oliveira, Cleiton Ferreira, Guilherme Morgado Runco

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was created by the rehabilitation team of the Hospital Universitario Pedro Ernesto  for patients undergoing total knee arthroplasty surgery. It contains guidelines and exercises to be performed in the immediate postoperative period in the nursery and after hospital discharge.</div></div>

[STEPS]
TITLE: Preparation of JF dye for retro-orbital injection in mice
AUTHORS: Boaz Mohar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>How to prepare </span><a href="https://www.janelia.org/open-science/janelia-fluor-dyes" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">JF dyes</span></a><span> for retro orbital injection.</span></div></div>

[STEPS]
?. [Dye preperation]
Take an aliqote with   of dye from  storage.
-20 °C
?. [Dye preperation]
Add   DMSO and do ~10 up and down with the pipeter
20 µl
?. [Dye preperation]
Add 20ul Pluronic F-127 20% in DMSO ~10 up and down with the pipeter
?. [Dye preperation]
Add 200ul 1x PBS ~10 up and down with the pipeter
?. [Dye preperation]
Drew 200ul without bubbles to a 29G syringe
?. [Dye preperation]
Follow the Retro-orbital injection protocol
{"blocks":[{"key":"cvbfu","text":"This protocol is following:","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"6srpk","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"38tk6","text":" ","type":"atomic","depth":0,"inlineStyleRanges":[],"entityRanges":[{"offset":0,"length":1,"key":0}],"data":[]},{"key":"5cbgj","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"151rj","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"cm638","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"9n4gq","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"6jfti","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[{"type":"citation","data":{"id":74,"guid":"8D511EE0AEDD11E9989AEB8A1843E6DC","align":null,"title":"Retro-orbital injections in mice","journal":"LabAnimal","authors":"Tal Yardeni, Michael Eckhaus, H. Douglas Morris, Marjan Huizing, Shelley Hoogstraten-Miller","doi":"10.1038\/laban0511-155","date":"1303303500","can_edit":1,"protocol_name":"Estimation of small molecule dye delivery to the brain of C57\/Bl mice","step_number":"5","step_guid":"C987F740AEDC11E9989AEB8A1843E6DC"}}]}
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Developing a Titration Kill Curve (G418, Hygromycin B and Puromycin)

[BEFORE_START]
Each mammalian cell line has a different sensitivity. Before experimentation, you should determine the optimal concentration of your antibiotic by developing the Titration Kill Curve for your cells.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Hydra Dissociation/Reaggregation 
AUTHORS: Yashodara Abeykoon, Adrienne Cho, Celina Juliano

[STEPS]
?. [Dissociation of Hydra]
Wash 30 Hydra several times in fresh Hydra Medium (HM) to remove debris
?. [Dissociation of Hydra]
Transfer Hydra to 1.5mL Eppendorf, remove media, and add 1mL 4°C Dissociation Medium (DM)
?. [Dissociation of Hydra]
Incubate Hydra at 4°C for 30-60 min (can incubate several hours without obvious detriment/benefit)
?. [Dissociation of Hydra]
Resuspend Hydra in 1mL fresh 4°C DM
?. [Dissociation of Hydra]
Shear by glass Pasteur pipette ~10-15x until supernatant becomes slightly turbid, allow Hydra to settle, and discard supernatant [ST1- refer to guidelines]
?. [Dissociation of Hydra]
Add 1mL fresh 4°C DM and shear by pipetting up and down with glass pasteur pipette ~1-1.5 min until solution becomes turbid, and approximately half of the available tissue is dissociated [ST2 - refer to guidelines]
?. [Dissociation of Hydra]
Let remaining large pieces settle. The supernatant contains the cells that will be collected and used for reaggregation
?. [Generating aggregates capable of developing into whole animals:]
Aliquot supernatant (dissociated cell suspension from step 7) to new Eppendorf tubes at an approximate desired cell density [ST3 -refer to guidelines]
?. [Generating aggregates capable of developing into whole animals:]
Centrifuge aliquots in microcentrifuge at 800g for 6 min [ST4 -refer to guidelines]
?. [Generating aggregates capable of developing into whole animals:]
Invert tubes for ~5-10 min to allow pellet to passively separate from the tube wall [ST5 -refer to guidelines]
?. [Generating aggregates capable of developing into whole animals:]
Transfer pellet to a 24-well plate containing a 50/50 mixture of DM and HM+Penicillin/Streptomycin (P/S) or 100% DM [ST6 -refer to guidelines]
?. [Generating aggregates capable of developing into whole animals:]
Periodically during development, replace the medium with HM+P/S
?. [Generating aggregates capable of developing into whole animals:]
If starting from 50/50 DM/HM+P/S, depending on the duration of the experiment, make the first medium exchange by 12hpa or 24hpa,  and make subsequent exchanges at ~24h intervals for the duration of the experiment.
?. [Generating aggregates capable of developing into whole animals:]
If starting with 100% DM, exchange into 50/50 DM/HM+P/S  at ~4 hours post aggregation (hpa), exhange into HM+P/S 100% at ~16hpa, and then continue to exange medium every ~24 hours  [ST7 - refer to guidelines]
?. [Generating aggregates capable of developing into whole animals:]
Enjoy your shiny, new Hydra
TITLE: Chicken immunization with KLH-gp120 fragment (254-274) conjugate.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Chicken immunization with peptides is inefective if only just the peptides are being inoculated. However, to make the immune response effective the fragment 254-274 of HIV-1 was conjugated with a carrier protein (KLH) that produced a critical immune response, assessed by ELISA, Immunoblot analysis and dot blot [1-4].  The Polson method (1990) can be used effectively to separate the IgY antibody from the egg yolk of immunized chickens [5].</div><div class = "text-block">References</div><div class = "text-block">1. Schade R, Pfister C, Italatsch R, Henklien P (1991) Polyclonal antibodies from chicken egg yolk-an alternative to the production of mammalian IgG type antibodies in rabbits. ATLA 19: 403-419. </div><div class = "text-block">2. Justiz Vaillant AA, Akpaka PE, Smikle M, McFarlane-Anderson N (2012) In vitro Inhibition of Staphylococcus aureus Isolates by Anti-Anti-Idiotypic Antibodies to Staphylococcal Protein (SpA). J Vaccines Vaccin 3: 127-131.</div><div class = "text-block">3.  Collins AM, Roberton DM, Hosking CS, Flannery GR (1991) Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies. Immunology 73: 388-393. </div><div class = "text-block">4.  Ho DD, Kaplan JC, Rackauskas IE, Gurney ME (1988) Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization. Science 239: 1021-1023. </div><div class = "text-block">5.  Polson A (1990) Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunol Invest 19: 253-258.</div></div>

[STEPS]
?. Two healthy layer chickens (brown Leghorn), aged approximately 6 months, are injected intramuscularly at multiple sites on the breast with the peptide-keyhole limpet hemocyanin (KLH) conjugate.
?. The chickens are immunized on day 0, with 0.2 µmol/ml of the fragment 254-274 of HIV gp120-conjugated to KLH (immunogen) in 0.5 ml complete Freund’s adjuvant (Sigma-Aldrich Co, St. Louis Missouri).
?. On days 15, 60, and 90 chickens are immunized with 0.2 µmol/ml of the immunogen in 0.5 ml incomplete Freund’s adjuvant (Sigma-Aldrich Co, St. Louis Missouri).
?. The eggs are collected post-immunization.The immunoglobulin Y is separated using the Polson method (1990).
TITLE: In vitro phosphorylation
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
In vitro phosphorylation

[STEPS]
1. 5 μg of α-syn GST fusion protein on beads were
resuspended in a buffer containing 20 mM HEPES, 1 mM ATP, 10 mM MgCl2,
2 mM DTT, pH 7.4. 



2. The reaction was initiated by adding 1 ug of human
recombinant PLK2 (Abcam, Cat#ab102108) and incubating at 30 °C with orbital
shaking overnight (Eppendorf ThermoMixer F1.5). 
3. Samples were then analyzed by
NuPAGE and immunoblotted or alternatively analyzed by LC-MS/MS
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in "<a href="http://protocols.io/view/FASP-Kit-Protocol-ORNL-Developed-for-Bacteriophage-ddn25d" target="_blank">FASP Kit Protocol-OrNL Developed for Bacteriophage</a>"

[STEPS]
?. 
?. 
?. 
?.