eamag/protocols-with-steps huggingface dataset:
TITLE: Prima UK--Reviews-2022
AUTHORS: prima  

[DESCRIPTION]
Prima UK--Reviews-2022

[STEPS]
SECTION: How does Prima UK Function?
1. hat's going on here?
The Prima Keto UK or "keto" diet is a low-carb, fat-rich eating plan that has been utilized for a really long time to treat explicit ailments. In the nineteenth 100 years, the Prima Keto UK was generally used to assist with controlling diabetes. In 1920 it was presented as a compelling treatment for epilepsy in youngsters in whom drug was ineffectual. The Prima Keto UK has likewise been tried and utilized in firmly observed settings for malignant growth, diabetes, polycystic ovary disorder, and Alzheimer's infection.

Notwithstanding, this diet is acquiring extensive consideration as a potential weight reduction system because of the low-carb diet frenzy, what began during the 1970s with the Atkins diet (an exceptionally low-carb, high-protein diet, which was a business achievement and promoted low-carb diets to another level). Today, other low-carb slims down including the Paleo, South Beach, and Dukan eats less carbs are largely high in protein yet moderate in fat. Interestingly, the Prima Keto UK is particular for its astoundingly high-fat substance, commonly 70% to 80%, however with just a moderate admission of protein.

How It Works
The reason of the Prima Keto UK for weight reduction is that assuming you deny the assemblage of glucose — the primary wellspring of energy for all phones in the body, which is acquired by eating carb food sources — an elective fuel called ketones is created from put away fat (along these lines, the expression "keto"- genic). The cerebrum requests the most glucose in a consistent inventory, around 120 grams everyday, in light of the fact that it can't store glucose.

 During fasting, or when very little carb is eaten, the body first pulls put away glucose from the liver and briefly separates muscle to deliver glucose. Assuming that this go on for 3-4 days and put away glucose is completely exhausted, blood levels of a chemical called insulin decline, and the body starts to involve fat as its essential fuel. The liver produces ketone bodies from fat, which can be utilized without a trace of glucose. [1]

Whenever ketone bodies collect in the blood, this is called ketosis. Sound people normally experience gentle ketosis during times of fasting (e.g., dozing for the time being) and exceptionally difficult activity. Defenders of the Prima Keto UK express that assuming the eating routine is painstakingly followed,Prima UK
https://www.jpost.com/promocontent/prima-keto-uk-united-kingdom-is-it-scam-or-trusted-706402
https://www.jpost.com/promocontent/prima-weight-loss-pills-uk-most-popular-pills-tablets-in-united-kingdom-704673
https://www.jpost.com/promocontent/prima-weight-loss-pills-uk-is-prima-diet-capsule-legitimate-or-scam-703262
https://www.mynewsdesk.com/health-news-corp/pressreleases/prima-weight-loss-pills-uk-everything-to-know-about-it-3175969
https://www.mynewsdesk.com/health-news-corp/pressreleases/prima-keto-weight-loss-united-kingdom-a-healthy-solution-for-your-weight-loss-journey-3181932
TITLE: allele.variability
AUTHORS: Sara Beier, Sara Beier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A prerequisite to improve the predictability of microbial community dynamics is to understand their assembly mechanisms. To study factors that contribute to microbial community assembly, we examined temporal dynamics of genes in five aquatic metagenome time series, originating from marine off-shore or coastal sites and one lake, while focusing on a trait-based data evaluation.</div><div class = "text-block">We expected to find gene-specific patterns for the temporal allele variability depending on the metacommunity size of carrier-taxa and variability of the milieu and the substrates that the resulting enzymes are exposed to. In more detail we hypothesized that a larger metacommunity would cause increased temporal variability of functional units, as shown previously for taxonomic units. Furthermore, we hypothesized that multi-copy genes feature higher temporal variability then single-copy genes, because gene multiplication is often the consequence of increased variability in (subtil) changes of substrate quality and quantity. Finally, we hypothesized that direct exposure of proteins to the extracellular environment would result in increased temporal variability of the respective gene compared to intracellular proteins as they would be exposed to highly variable conditions. The first two hypotheses were confirmed in all, while an effect of the subcellular location of gene-products was only seen in three out of the five time series. </div><div class = "text-block">The gene with highest allele variability throughout all datasets was an iron transporter, which also represents a target for phage infections. This finding points to the general importance of iron transporter mediated phage infections on the assembly and maintenance of diversity of aquatic prokaryotes.</div></div>

[STEPS]
?. removal of nextera adaptors (cutadapt v1.8.3)
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TITLE: AMPureXP purification
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
PCR-product purification with AMPureXP solution.
 
From the manual:
"The Agencourt AMPure XP system is a highly efficient, easily automated PCR purification system that delivers superior-quality DNA with no salt carryover. Requiring no centrifugation or filtration, Agencourt AMPure XP can be easily used in manual and automated 96- or 384-well formats."
 
High recovery of amplicons greater than 100 bp
Efficient removal of unincorporated dNTPs, primers, primer dimers, salts and other contaminants
Stable PCR products post-cleanup: No PCR degradation after storage at 4° C for seven days
Efficient recovery of double-stranded and single-stranded DNA templates
Consistent recovery throughout the kit’s 12-month shelf life
Faster manual and automated processing as compared to traditional post-PCR cleanup methods
 

[STEPS]
1. Shake the Agencourt AMPureXP bottle to fully resuspend magnetic particles.
2. Add sample Vol µL × 1.8 (or less i.e. 0.8) of the Agencourt AMPure XP solution. Pipette mix 10 times, or vortex a few seconds.
3. Incubate at room temperature for 5 minutes.
5 min
4. Place the reaction plate/tube onto an Agencourt SPRIPlate Super Magnet Plate for 2 minutes to separate beads from solution.
2 min
5. Aspirate the supernatant from the reaction plate/tube and discard.
6. Dispense 200 µL of 70% ethanol and incubate at room temperature for at least 30 seconds. Aspirate out the ethanol and discard. Repeat for a total of two washes.
200 1
30 s
7. Leave plate to dry for ca. 15-20 min. at room temperature.
15 min
8. Add at 40 µL elution buffer (10 mM Tris buffer, no TE!!) Volume can be lower, in the past even 20µL worked, but then it is harder to get the purified sample out of the plate/tube. Pipette mix 10 times or vortex a few seconds.
40 1
9. Incubate at room temperature for 2 minutes.
2 min
10. Place the reaction plate onto an Agencourt SPRIPlate Super Magnet Plate for 1 minute to seperate beads from solution.
1 min
11. Transfer purified product to a fresh plate or PCR tube (better than 1.5mL tubes, as you can use a multichannel pipette for quantification!).
TITLE: Investigation_of_mitophagy_in_Hippo_neurons
AUTHORS: OLIVIA  HARDING, Chantell S. Evans

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We developed a method for assessing mitochondrial clearance in primary hippocampal neurons. </div></div>

[STEPS]
?. [Plating and maintenance of hippocampal neurons]
Plate primary hippocampal Sprague Dawley rat neurons (embryonic day 18) on 35 mm glass bottomed dishes at 250,00 cells per dish in MEM supplemented with 10% horse serum, 33 mM D-glucose, and 1 mM sodium pyruvate.
?. [Plating and maintenance of hippocampal neurons]
Add AraC (5 uM) one day post-plating to prevent glial proliferation
?. [Plating and maintenance of hippocampal neurons]
Replace media with Neurobasal (Gibco) supplemented with 33 mM D-glucose, 2 mM GlutaMAX (Invitrogen), 100 units/ml penicillin, 100 ug/ml streptomycin, and 2% B-27 (ThermoFisher) (Maintenance Media; MM) and continue to maintain at 37 degrees C, 5% CO2.
?. [Plating and maintenance of hippocampal neurons]
Precoat dishes with 0.5 mg/mL poly-L-lysine.
?. [Transfection of primary neurons]
Transfect neurons 5 days after initial plating.
?. [Transfection of primary neurons]
Simultaneously transfect 0.8-1.2 ug total plasmid per 35-mm dish and 45 pmol siRNA. Use Lipofectamine 2000 Transfection Reagent (ThermoFisher).
?. [Transfection of primary neurons]
Incubate transfected neurons 36-48 hours.
?. [Mitophagy induction in neurons]
Replace neuron media with MM containing 3 nM Antimycin A (or, for control conditions, media containing no small molecule inhibitor) and incubate for 2 hours.
?. [Fluorescent labeling in neurons]
For Halo and/or SNAP tagged expressed proteins, incubate neurons in 100 nM Halo and/or 2 uM SNAP ligand of preferred colors for 30 min. Wash 2X and rest 30 min in conditioned media.
?. [Fluorescent labeling in neurons]
To measure mitochondrial membrane potential, incubate neurons in 2.5 nM TMRE for 30 min
?. [Imaging neurons]
Replace MM with HibernateE (Brain Bits) supplemented with 2% B27 and 33 mM D-glucose and rest at least 15 min in imaging chamber of microscope.
?. [Imaging neurons]
Add Antimycin A to HibE media for treated conditions. Add TMRE to HibE media for mitochondrial potential measurements.
?. [Imaging neurons]
Image neurons, for example, with a Nikon Eclipse Ti Microscope with a 100X objective (Apochromat, 1.49-N.A. oil  immersion)  and  an  UltraView  Vox  spinning  disk  confocal  system  (PerkinElmer). Collect Z-stacks at 0.15 nm/step with Volocity acquisition software (PerkinElmer).
?. [Plating and maintenance of hippocampal neurons]
Incubate neurons for at least 2 but no more than 5 hours at 37 degrees C, 5% CO2.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Transformation of <em>Acanthamoeba castellanii </em>Neff (ATCC-30010) with plasmid DNA can be achieved with a modified version of the protocol described in Peng, Omaruddin, &amp; Bateman (2005) using constructs from Bateman (2010). </p>

[BEFORE_START]
<p>Materials needed:</p>
<p> </p>
<ul>
<li>PYG medium with additives (0.75% yeast extract, 0.75% proteose peptone, 2 mM KH<sub>2</sub>PO<sub>4</sub>, 1 mM MgSO<sub>4</sub>, 1.5% glucose, 0.1 mM ferric citrate, 0.05 mM CaCl<sub>2</sub>, 1 μg/mL thiamine, 0.2 μg/mL D-biotin, and 1 ng/mL vitamin B12.</li>
<li>Plasmid DNA</li>
<li>Qiagen SuperFect</li>
<li>Encystment medium (20 mM Tris–HCl [pH 8.8], 100 mM KCl, 8 mM MgSO<sub>4</sub>, 0.4 mM CaCl<sub>2</sub>, 1 mM NaHCO<sub>3</sub>)</li>
</ul>
<p> </p>

[STEPS]
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TITLE: Nissl Staining
AUTHORS: Pranay Srivastava, Xiqun Chen

[DESCRIPTION]
This protocol details the staining procedure and mounting procedure of Nissl staining.

[STEPS]
SECTION: Staining Procedure
1. Mount sections on positive charged plus slides. Air dry sections  .

SECTION: Staining Procedure
2. Incubate the slides directly into 1:1 ethanol/chloroform for 30 min.
(All the steps mentioned should be done under the hood)
SECTION: Staining Procedure
3. Incubate in 100% ethanol  2 min 

SECTION: Staining Procedure
8. Stain in 0.1% cresyl violet solution for 6 min.
 

SECTION: Staining Procedure
9. Rinse quickly in distilled water.
(check the slides for the best result under the microscope)
SECTION: Staining Procedure
10. Incubate in 50% ethanol (3 dips in the container)
SECTION: Staining Procedure
11. Incubate in 100% ethanol (3 dips in the container)
SECTION: Mounting Procedure
13. Pour ~3 mL Permount solutioninto a 5 mL tube and use a transfer pipette for mounting.

SECTION: Mounting Procedure
14. Remove one of the slides from the xylene with tweezer and dry by dabbing it on a paper towel. Then place down flat.
SECTION: Mounting Procedure
15. Put 6-7 drops of Permount along the middle of the slide.
SECTION: Mounting Procedure
16. Take a cover slip and place the left side down onto the slide.
 

SECTION: Mounting Procedure
17. Once the cover slip is completely on the slide, drain any extra permount on a paper towel and then lay the slides to dry.
SECTION: Mounting Procedure
18. If you make a mistake while cover slipping, put the slide with the cover slip back into xylene.
 

SECTION: Staining Procedure
10.1. Incubate in 95% ethanol (3 dips in the container)
SECTION: Staining Procedure
4. Incubate in 95% Ethanol2 min 

SECTION: Staining Procedure
5. Incubate in 70% Ethanol2 min 
SECTION: Staining Procedure
6. Incubate in 50% Ethanol2 min 
SECTION: Staining Procedure
7. Wash in distilled water for 5 min 

SECTION: Staining Procedure
12. Incubate in xylene 3 min
TITLE: SMARTerV4 (0.5x) Amplification for single-cell or single-nuclei RNASeq Protocol
AUTHORS: ZengU19 BRAIN grant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to generate full-length cDNA from single cells, or nuclei, using Takara SMARTer V4.</div></div>

[STEPS]
TITLE: UroMOCA and ColoMOCA implantation protocol  
AUTHORS: Brett Hanzlicek, Margot Damsaser, Dennis Bourbeau

[DESCRIPTION]
This protocol describes the implantation of our bladder (UroMOCA) and bowel device (ColoMOCA) in pigs.

[STEPS]
SECTION: Anesthesia
1. Anesthetize/prep the animal for surgery.

         Aseptic procedures by trained personnel are utilized during the surgery. All tools are sterilized                     prior to insertion. Drapes will be used and personnel performing surgery will wear clean surgical     scrubs, sterile gowns/gloves and masks.
SECTION: Laparotomy
5. Make a midline suprapubic incision ~9 cm long to expose the bladder, peritoneal cavity and viscera.
SECTION: Colotomy
12. Make a single, small incision into the colon.
SECTION: Insert and anchor ColoMOCA
13. Insert device through the small incision and secure in place with sterile non-absorbable sutures.
SECTION: Test Wireless Transmission
14. Wake the coloMOCA with radio if not already awake.  Confirm data transmission.
SECTION: Close incision
15. Close the colon with an absorbable suture in an interrupted pattern
Close the peritoneum and fascia with absorbable sutures using a continuous suture pattern.
Close the subcutaneous tissue and skin with non-absorbable monofilament suture with a cutting needle, using an interrupted suture pattern.Skin staples may also be used.
SECTION: Test Wireless Transmission (2)
16. Confirm data transmission from both the UroMOCA and ColoMOCA, and then put devices to sleep
SECTION: CT-Imaging
17. Image to verify placement of the sensors.
SECTION: Place jacket on the pig
18. Place and zip the jacket on the pig while still anesthetized.
SECTION: Wake up the animal
19. Stay with the animal until fully awake.  Return to pig room.
SECTION: Laparotomy
3. Place animal in supine position.
SECTION: Laparotomy
4. Administer analgesic at injection site.
SECTION: Anesthesia
2. Insert single lumen 8 Fr catheter, and drain bladder.  Save some urine for urinalysis.
SECTION: Laparotomy
6. Isolate bladder and incise the bladder dome.
SECTION: UroMOCA placement and bladder closure
7. Place UroMOCA device into bladder.
SECTION: UroMOCA placement and bladder closure
8. Close bladder incision and evaluate integrity.
SECTION: UroMOCA placement and bladder closure
9. Place sterile drape on abdomen for UroMOCA transmission coil.
SECTION: UroMOCA placement and bladder closure
10. Verify function of UroMOCA in vivo.
SECTION: Colotomy
11. Isolate the region of the colon where the implant will go.
TITLE:  DNA extraction and quantification 
AUTHORS: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">DNA extraction and quantification</span></div></div>

[STEPS]
?. Resuspend previously grown bacterial cells (approximately 50 mg of bacterial fresh biomass) in 400 µL de TE 1X (10mM of Tris-HCl, 1mM of EDTA pH 8,0).
?. Add 1 µL of lysozyme (100mg/ml) shortly vórtex, and incubate for 1 h at 37 °C.
?. Add 67,5 µL of SDS 10 % (pre-heated at 65 °C) and 2,5 µL of Proteinase K (10 mg/mL) and shortly vórtex, and incubate at 65 °C for 10 min.
?. Add 100 µL of NaCl at 5M and 100 µL of a solution of Cetyltrimethylammonium Bromide (CTAB)/NaCl (0,03 M/0,07 M), both pre-heated at 65 °C, and vortex until observing a whitish color and incubate at 65 °C for 10 min.
?. Add 750 µL of chloroform/isoamyl alcohol (24:1), vortex for 10 s and centrifugate at 12.000 rpm for 5 min.
?. Transfer the supernatant (approximately 800 µL) to a new tube and add 0,6 volumes of isopropyl alcohol to precipitate the DNA and gently invert the tube.
?. Store at -20 °C for 30-60 min in case precipitated DNA is not observed.
?. Centrifugate at 4 °C at 12000 rpm for 5 min.
?. Discard the supernatant and add 1 mL of ice-cold ethanol 70% (-20 °C).
?. Centrifugate at 12.000 rpm for 5 min and discard the supernatant.
?. Add 1 mL of ice-cold ethanol 100 % (-20 °C).
?. Centrifugate at 12.000 rpm for 5 min and discard the supernatant.
?. Centrifugate at 12.000 rpm at 4 °C for 1 min to let the ethanol evaporate at room temperature.
?. Invert the tubes, place them on absorbent paper, and then use a speedvac at 36 °C for 30 min to let them completely dry.
?. Resuspend the pellet in a volume of 20 µL of MilliQ water (add more water if necessary, according to the concentration of the pellet).
?. Store the DNA at -20 °C.
?. Check the DNA quality by migrating different concentrations of it and using a control λ phage concentration of 50 ng/µL in an agarose gel with 1 % of TBE 1X ran for 30 min at 5 V per cm2
TITLE: Traditional Fishing Knowledge and Fishing Policy Analysis
AUTHORS: Kai Bloom

[DESCRIPTION]
Examine how traditional fishing knowledge is not being passed down to the new generations of fishermen in Mo'orea, French Polynesia, thus causing fishing policy to be simplified. Additionally, examine the effects that simplified policy has on fish populations and if it further perpetuates the loss of traditional fishing knowledge. We expect to observe a reduction in fish populations after the implementation of simpler fishing policies, and that such policy, along with modern fishing techniques, are leading to increased loss of traditional fishing knowledge.

[BEFORE_START]
Consent is key, do not pry for information or persistently ask questions if the interviewee is hesitant or uncomfortable!  

[STEPS]
SECTION: Examining The Loss of Traditional Fishing Knowledge
1. Identify groups of local fishermen, both with traditional knowledge and new-generation fishermen
SECTION: Examining The Loss of Traditional Fishing Knowledge
3. Conduct interviews with fishermen
SECTION: Examining The Loss of Traditional Fishing Knowledge
4. Transcribe and document interview answers 
SECTION: Examining The Loss of Traditional Fishing Knowledge
3.1. Ask about traditional knowledge, what knowledge they possess, how it is passed down, and why it is dying out.
SECTION: Examining The Loss of Traditional Fishing Knowledge
2. Build trust and establish a relationship with the fishermen by working and interacting with them consistently
SECTION: Examining The Loss of Traditional Fishing Knowledge
3.2. Ask about modern fishing policy: is it helpful? Do they agree with the management strategies? What changes would they like to see?  
SECTION: Examining The Impact on Fish Populations
5. Gather existing information about fish populations around Mo'orea from published datasets
SECTION: Examining The Impact on Fish Populations
6. Cross-reference fish population data with historical fishing policy and marine management changes
SECTION: Examining The Impact on Fish Populations
7. Note key changes and determine if there was a significant impact on fish populations during that time
TITLE: Baermann Funnel Extraction
AUTHORS: Casey A Schlenker

[DESCRIPTION]
Baermann Funnel Extraction

[STEPS]
SECTION: Sample Preparation
1. Sieve soil to 2mm
SECTION: Sample Preparation
2. Weigh out 50 g in a weigh boat

SECTION: Sample Preparation
3. Place tissue paper on top of Baermann funnel apparatus
SECTION: Sample Preparation
4. Place weighed and sieved soil on top of filter paper
SECTION: Sample Preparation
5. Fill Baermann funnel with DI water using a DI water bottle so that soil sample is saturated but not submerged in water
SECTION: Sample Preparation
6. Cover all Baermann funnels with a plastic Uline 
SECTION: Sample Preparation
7. At 1440 min and 2880 min mark, check soil and refill any funnels with decreased water levels to prevent the soil from drying out.

SECTION: Sample Preparation
8. At 4320 min empty funnel over a 25um mesh sieve to collect nematodes

SECTION: Sample Preparation
9. Transfer the nematodes that are on the sieve into a labelled centrifuge tube using a hot fixative
 

SECTION: Sample Preparation
11. Count the entire sample at 50x magnification using a dissecting scope
SECTION: Sample Preparation
12. Identify 100 nematodes
SECTION: Sample Preparation
10. Collect soil from top of Baermann funnel; dry and weigh to get gravimetric soil moisture content
TITLE: SJB Artificial Seawater Medium Protocol
AUTHORS: Shelby J Barnes, Michael Henson, Cameron Thrash

[DESCRIPTION]
SJB Artificial Seawater Medium Protocol

SJB Artificial Seawater Medium is an amended version of JW and MWH generations of Thrash Lab artificial seawater medium. 



[STEPS]
SECTION: Stock Preparation
1. Prepare the following stocks prior to medium assembly. 



SECTION: Base Salts
2. Using an acid-washed and autoclaved one Liter screw-top Pyrex bottle, combine basic salts in 997.4095 mL MilliQ-filtered water. (stock volumes subtracted)
SECTION: Stock Addition
3. Stocks should be added in a Biosafety cabinet or hood with appropriate ventilation. 


Swirl or invert media between stock additions. 
SECTION: Base Salts
2.1. Mix continuously with stir bar or invert the tightly capped bottle. 

Each chemical should be fully dissolved before next addition. 
SECTION: Stock Addition
3.1. Add (per Liter): 
10 μL Trace Metal Mix
10µL Vitamin Mix
20 µL Amino Acid Mix 
50 µL Misc. Mix 
500 µL Inorganic Nitrogen Mix
0.5 µL Fatty Acid Mix
1mL Iron Mix 
1mL Phosphate Mix
SECTION: Stock Preparation
1.1. Iron mix (1,000x)
Trace Metals (100,000x) 
Vitamins (100,000x) 
AA mix (500x) 
Misc. mix (20,000x)
Phosphate mix (1000x) 
Inorganic Nitrogen mix (2,000x) 
Fatty Acid mix (2,000,000x) 
SECTION: Stock Addition
4. When in the biosafety cabinet or laminar fume hood filter-sterilize medium using a 0.2 µm filter into another sterilized one Liter Pyrex bottle.
SECTION: Stock Addition
5. pH and record.

Media can be stored at room temp (~25 C°) in the tightly closed Pyrex bottle. 
SECTION: Stock Preparation
1.2. Filter sterilize stocks using 0.2 µM PES filter.