eamag/protocols-with-steps huggingface dataset:
TITLE: Oprah Winfrey Keto Pills Reviews - Where To Buy It?
AUTHORS: oprahwinfreypillsreviews  

[DESCRIPTION]
Oprah Winfrey Keto Pills Reviews - Where To Buy It?


[STEPS]
1. Oprah Winfrey Keto Pills Reviews - Where To Buy It?
Oprah Winfrey Keto Reviews-If you have decided that the time has come to lose a many redundant pounds, know that the path to take is neither easy nor short.
Still, presumably, with the right provocation, If it were enough to suffer exhausting sessions in the spa or confined low-calorie administrations. 
Losing weight, still, is the result of multiple measures, constant commitment, and great provocation. 
Losing redundant pounds means reversing the course; combining a controlled diet with a profitable physical exertion, the energy expenditure exceeds the calories introduced and we begin to lose weight. 
Among the multitudinous salutary administrations in rotation, one of the best known and most effective is really the Keto Diet which is gaining wide acceptance among the public with positive feedback also from assiduity experts. 
Faced with the absence of ingested carbohydrates, the body produces glucose, essential for survival, and begins to affect the reserves of fat to produce energy. In this way, a slimming process begins which affects only the fat pads and not the muscle mass. 
The Keto Diet is extensively used in the field of salutary strategies aimed at reducing weight for substantially aesthetic purposes, but it's also used to combat some pathologies ( similar as some epileptic forms) and forms of severe rotundity. 
Of course, the Keto Diet can be tiring for the body especially during the maximum moment of product of ketogenic bodies; for this reason, it's judicious to combine the chosen diet with a food supplement able of enhancing the salutary goods of the diet and neutralizing the general sense of fatigue. 
So let’s find out what Oprah Winfrey Keto is, its main characteristics, and what its fabulous action is. 
WHAT IS OPRAH WINFREY KETO – FULL REVIEWS 
Veritably analogous to the Oprah Winfrey Keto food supplement, it's a fully natural supplement whose abecedarian purpose is to enhance the salutary goods of the Keto diet and to combat the side goods that can do including lack of energy, somnolence, headache, and gastrointestinal diseases. 
The active constituents present in the supplement are 
Vitamins of group B 
Potassium 
Magnesium 
Glutamine 
Official Website:- https://freaknewswire.com/index.php/2022/05/13/oprah-winfrey-keto-pills-reviews-price-benefits/
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This in silico protocol allows to have an insight into the sequence divergence of selected genomic region, including missing information, gaps and allignment with Open Reading Frames. The protocol is written for model plant Arabidopsis thaliana, but can be possibly adapted to plotting the divergence of any organism, for which multiple accessions were resequenced. We used this script to produce divergence plots in our paper

Magdalena M. Julkowska, Karlijn Klei, Like Fokkens, Michel A. Haring, M. Eric Schranz, Christa Testerink; Natural variation in rosette size under salt stress conditions corresponds to developmental differences between Arabidopsis accessions and allelic variation in theLRR-KISSgene,Journal of Experimental Botany, Volume 67, Issue 8, 1 April 2016, Pages 2127–2138,https://doi.org/10.1093/jxb/erw015

[BEFORE_START]
 Download gnuplot (http://www.gnuplot.info/download.html) and install it by putting the download folder into your “Applications”
 Download ClustalO (http://www.clustal.org/omega/) and install it by putting the download folder into your “Applications”

You can best do the installation by using HomeBrew package by typing the following command in your terminal window:

a
Once you have the Homebrew installed, you can install packages by typing in your terminal following command:

a


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TITLE: Effective asexual propagation (fragmentation) method of the soft coral Xenia umbellata
AUTHORS: Sohyoung   Kim, Christian Wild, Arjen Tilstra

[DESCRIPTION]
Many asexual propagation methods for hard corals can easily be found in scientific papers, while methods for soft corals are mostly present in grey literature or aquarium hobbyists’ forums. Moreover, fragmentation methods for hard corals are difficult to be adopted by many soft corals since they lack a rigid support system. Even though some soft corals may be considered as easy and fast growers, it is difficult when it comes to preparing healthy fragments in large quantities to use for experiments. Thus, the Plug mesh method was formulated to easily fragment one soft coral type lacking any rigid support system, the Xenia umbellata, efficiently. This method was coined by modifying a method named theTunnel cube method developed by Curry (2020) and could be used for many other soft corals with similar characteristics. 

[BEFORE_START]
  

Prepare two eggcrates of the same size. 
Prepare the needed number of coral plugs. Make sure they are not placed right beside each other but being adjacent diagonally is ok.
Cut a mesh fabric to fit the selected eggcrate size. Use a mesh with large enough holes for water to flow while small enough to keep fragments inside. A diameter of 3.5mm or larger is recommended. 
Secure the eggcrates and coral plugs with rubber bands. 
Prepare extra rubber bands to secure the top mesh fabric after coral fragmentation.
If fragments will be placed in a nursery tank, make sure to prepare the nursery tank to have ambient water flow allowing sufficient circulation within the tank.

[STEPS]
1. Select a coral stock and prepare it in a water basin filled with identical water from where it comes from. 

  

2. Cut prepared stock into the desired size with scissors or a scalpel. Use tweezers if needed to secure them and always keep them in the water.

  

3. Gently allocate them on top of plugs inside eggcrate grids.

  

4. Cover the top of the eggcrate with prepared mesh and rubber bands and then put the assembly into the tank.

  

5. Check every 24 hours to make sure that they have attached to the plug, not the wall of the eggcrate.

  

6. If they have attached to the eggcrate wall, gently scrape them off and place the fragment back on top of the plug.
7. Once the fragments are firmly attached to plugs (after 24~72hours), they can be removed from the eggcrate assembly. Firm attachment can be assessed by gently shaking the plug.
TITLE: Transfectionof constructs in diplonemids to block NHEJ pathway using the inhibitors of KU70/80 proteins.
AUTHORS: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2  and Julius Lukeš1, 2, 3

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The process of NHEJ is completed when the DNA ligase complex ligates the broken ends of the DNA. Ku70-Ku80 are the DNA-dependent protein kinase heterodimeric regulatory factor that forms a link between two broken DNA ends and structurally support, align and protect them from further degradation. W7 inhibitor (W7(N-(6-aminohexyl)-5-chloro1-naphthalenesulfonamide) inhibits the cofactor of inositol hexakisphophate which effects the regulatory factor of Ku heterodimers.</div></div>

[STEPS]
?. Step 0: Pre-incubation of Diplonema cells for 4 hours with W7 (5 mg/ml) beforeelectroporation. EC value of W7 was calculated by Alamar blue assay
?. Count the cells  and plan to 1 nucleofection with 5x 107 cells for each construct.
?. Resuspendthe cell pellet in 100ul of AMAXA Human T- cell solution at 4C (from refrigerator combine 81.8ul of Human T-cell nucleofectorsolution + 18.2ul Supplement).
?. Harvest thecells by centrifugation at 1300xg for 5 min at room temperature in Swing BucketRotor
?. Add 5-10ug of (PCR) (linearized DNA) into the cuvette(resuspend in 10ul of H2O).
?. : Put everything into the cuvette, close the cap and place in the electroporator, cuvette should only fit in one direction, but metal sides should face towards you.
?. Press for the Program X-001 to electroporate.
?. Transfer the entire cell- DNA transfectant into 10ml of Diplonema media (chloramphenicol) with the supplied disposable micropipettes
?. Immediately observe the flasks containing transfectants under a microscopeand place them in the incubator at 27C + shaker and let them grow for 6-8h.
?. After 6-8 hours took out the flasks and make controls which should be without antibiotics - put 1ml of the electroporated cells in the first well of 24 well plates.
?. In the rest - 9ml -transfectants add Puromycin antibiotics in the flasks.
?. Add 1.5ml of electroporated cells in the first row(6 wells) of 24 well plates. Put 900ul of the media (+ selectable drug) in the rest of the wells. Take 100ul from first well and transfer to the next to make 10x serial dilution and so on...up to the lowest concentration 5 x 102 cells/ml and let them grow untill selection of clones is done.
?. Result: Unfortunately, targeting to the planned position (N-terminal tagging of alpha-tubulin with mCherry under puromycinR selection) did not work in any of the obtained clones.
TITLE: Primers for bark beetles and associated fungi
AUTHORS: Andrew J. Johnson, Demian F Gomez, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the primers used for bark beetles and associated fungi.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The microbiological testing method for Salmonella spp. as used for testing primary envrionmental samples collected from poultry environments</p>
<p>The method is described as detailed in accordance with the Australian Standard 5013.10-2009 Horizontal method for the detection of <em>Salmonella</em> spp. (ISO 6579:2002, MOD).</p>
<p>This method is a modification of the ISO 6579:2002  Horizontal method for the detection of <em>Salmonella</em> spp.. The difference in methodology is due to the use of a different control isolate.</p>

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TITLE: Relative quantification of mRNA transcript levels by qPCR
AUTHORS: Will Hancock-Cerutti, Zheng Wu, Gerald S. Shadel, Pietro De Camilli

[DESCRIPTION]
This method describes isolation of RNA from cultured cells, generation of cDNA, and relative quantification of transcript levels by qPCR.

[STEPS]
SECTION: Cell culture and treatments
1. Culture the HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 Milimolar (mM) L-glutamine (all from Gibco).

SECTION: Cell culture and treatments
2. For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.
SECTION: Cell culture and treatments
3. For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells 4320 min after siRNA transfection.

SECTION: Cell lysis, RNA purification, and qPCR
4. Aspirate media from cells and rinse cells with PBS on ice.

SECTION: Cell lysis, RNA purification, and qPCR
5. Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.
SECTION: Cell lysis, RNA purification, and qPCR
6. Generate cDNA from 1 µg purified RNA using iScript cDNA synthesis Kit (Bio-Rad) according to manufacturer’s protocol.

SECTION: Cell lysis, RNA purification, and qPCR
7. Dilute the iScript reaction to a total of 400 µL Sterile Water (American Bio).

SECTION: Cell lysis, RNA purification, and qPCR
8. Combine 10 µL SYBR Green Master Mix (BioRad) with 6.78 µL Sterile Water (American Bio) per sample.

SECTION: Cell lysis, RNA purification, and qPCR
9. Combine 16.78 µL diluted SYBR Green Master Mix with 0.61 µL each of 10 Micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate. Perform at least two technical replicates for each sample.

SECTION: Cell lysis, RNA purification, and qPCR
10. Pipette 2 µL of diluted RNA from step 7 in well with SYBR Green Master Mix.

SECTION: Cell lysis, RNA purification, and qPCR
11. Cover plate with Optical Adhesive Covers (Applied Biosystems).
SECTION: Cell lysis, RNA purification, and qPCR
12. Spin down plate in table top centrifuge.
SECTION: Cell lysis, RNA purification, and qPCR
13. Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:
 
          95 ˚C   3 min      95 ˚C   10 sec   Repeat 39x    55 ˚C   10 sec      72˚C   30 sec      95 ˚C   10 sec      65 ˚C   5 sec      95 ˚C   5 sec    
 
SECTION: Data analysis
14. Subtract the housekeeping gene (b-actin) mean threshhold cycle (Ct) values from transcript of interest mean Ct values to calculate ΔCt.
SECTION: Data analysis
15. Subtract the ΔCt of the control sample from each sample ΔCt to calculate the ΔΔCt value.
SECTION: Data analysis
16. Calculate relative expression using the 2−ΔΔCt method.
TITLE: Brain Clearing (iDISCO)
AUTHORS: Adam Kimbrough, Olivier George

[STEPS]
?. Day 1: See materials for solutions to mix:Dehydrate brains with methanol H2O series 1h each wash increasing in concentration (20%, 40%, 60%, 80%, 100%) and once again with 100% (6h total)Incubate brains in 66% dichloromethane (DCM)/33% methanol overnightDay 2:Wash the brains twice for 1h each with 100% methanol to rinse out DCM and then chill samples for 10 minutes at 4 CChill 5% hydrogen peroxide in methanol and then incubate the samples overnight at 4 C in the solutionDay 3:Rehydrate the samples using decreasing concentrations of methanol (1h each): 80%, 60%, 40%, 20%, PBS (5h total)Wash in Ptx.2 twice for 1h (2h total)Incubate samples in Permeablization solution overnight at 37 CDay 4:Leave samples in Permeabilization solution at 37 CDay 5:Incubate samples in Blocking solution at 37 CDay 6:Leave samples in Blocking solution at 37 CDay 7:Incubate samples in primary antibody (based on what antibody is being used) mixed with 5% DMSO, 3% normal donkey serum in PtwH for 7 days at 37CDay 14:Wash samples with PtwH 5x (1h minimum per wash) and leave in the last wash overnight (room temperature)Day 15: Samples covered with foil from this point forwardIncubate samples in secondary antibody (matched to primary) mixed with 3% normal donkey serum in PtwH for 7 days at 37 CDay 22:Wash samples with PtwH 5x (1h minimum per wash) and leave in the last wash overnight (room temperature)Day 23:Dehydrate brains with methanol H2O series 1h each wash increasing in concentration (20%, 40%, 60%, 80%, 100%).Incubate in a fresh solution of 100% methanol and leave samples overnightDay 24:Incubate samples for 3h in 66% DCM/33% MethanolIncubate samples for 15 minutes 2x in 100% DCM to clear off any methanol residueMove samples to Dibenzyl Ether without shaking for long term storage Store at room temperature in the dark until imaged
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Short DNA probes, for example, those used in characterizing protein-DNA complexes, have historically been radiolabeled with <sup>32</sup>P to allow their detection and quantitation following native polyacrylamide gel electrophoresis (PAGE). For reasons of economy or safety, alternative means of DNA detection need to be used. Described here is the resolution of 5' IRD700 and IRD800 end-labeled DNAs by native PAGE and their visualization and quantitation by IR fluorescence imaging.</p>

[BEFORE_START]
<p>Stock reagents used:</p>
<p> </p>
<p><em>5× Tris-borate EDTA (TBE) buffer</em></p>
<p>27 g Tris base</p>
<p>13.8 g Boric Acid</p>
<p>1.86 g EDTA•Na<sub>2</sub>•2 H<sub>2</sub>O</p>
<p>Weigh separately. Dissolve with stirring in 400 mL deionized water. Dilute to 500 mL final. Store at 4 ºC in a tightly capped 500 mL glass bottle. Routinely stable for over 2 months. The appearance of an accumulating white precipitate indicates that TBE buffer is no longer suitable for use.</p>
<p> </p>
<p><em>40% (19:1) Acrylamide </em></p>
<p>19 g acrylamide</p>
<p>1 g bisacrylamide</p>
<p>Weigh separately. Dissolve with stirring in 40 mL deionized water. Dilute to 50 mL final. Store at 4 ºC in a tightly capped 50 mL plastic centrifuge tube. Routinely stable for over 2 months. </p>
<p> </p>
<p><em>10% Ammonium persulfate </em></p>
<p>Weigh approximately 100 mg ammonium persulfate in a 1.5 mL microcentrifuge tube. Add 9× measured weight in deionized water (approx. 900 μL). Vortex to dissolve. Store at 4 ºC, tightly capped. Routinely stable for up to 1 month.</p>
<p> </p>
<p><em>6× Native Orange Loading Dye</em></p>
<p>2 g glucose</p>
<p>90 mg Orange G</p>
<p>Weigh separately. Dissolve with stirring in 8 mL deionized water. Dilute to 10 mL final. Aliquot into ten 1.5 mL microcentrifuge tubes. Store at –20 ºC. Stable indefinitely. Thaw and vortex to mix thoroughly before use.</p>
<p> </p>
<p> </p>
<p> </p>

[GUIDELINES]
<p>Protocol development supported by grants from the NIH (<a href="&quot;https://projectreporter.nih.gov/project_info_description.cfm?a" target="_blank">1R15GM104833-01</a>) and NSF (<a href="https://www.nsf.gov/awardsearch/showAward?AWD_ID=1714778&amp;HistoricalAwards=false" target="_blank">MCB-1714778</a>).</p>

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TITLE: Subcutaneous Injection Adult Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes general procedures used for subcutaneous injection in adult mice.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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TITLE: Donor Acceptance Criteria for TMC Florida/Zurich HuBMAP Inclusion
AUTHORS: Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This document outlines the required criteria for inclusion for donors of thymus, lymph nodes and spleen in the HuBMAP project.</div></div>

[STEPS]
?. [Donor Criteria]
Donor must be less than seven days on ventilator
?. [Donor Criteria]
Donor tissue must be received within 24 hours of recovery
?. [Donor Criteria]
Contraindications for donor acceptance are
?. [Donor Criteria]
More than 4 blood transfusions
?. [Donor Criteria]
Any autoimmune diseases
?. [Donor Criteria]
Chromosomal abnormalities
?. [Donor Criteria]
Bacterial or viral meningitis or MRSA in any culture
?. [Donor Criteria]
Sepsis
?. [Donor Criteria]
Observable infarction, rupture, deflation or gross abnormality
?. [Spleen Specific Criterion]
Must receive at least 60% of the intact spleen, including hilum.
?. [Spleen Specific Criterion]
No spenomegly
?. [Donor Criteria]
BMI must be in keeping wioth CDC published growth charts 5th to 95th percentile
?. [Lymph Node Specifc Criterion]
Must receive at least 10 intact lymph nodes
?. [Thymus Specific Criterion]
Must be under the age of 50 to permit for adeqquate lymphoid tissue presence
?. [Donor Criteria]
A copy of the complete recovery checklist must be provided
TITLE: BAF_S07_SpeedVac SPD1030
AUTHORS: Nicholas Sherman

[DESCRIPTION]
General use of the SpeedVac for drying samples that contain aqueous or organic solvents. This is room temp and does not keep samples frozen like lyophilization.

[STEPS]
SECTION: SpeedVac SPD1030 ThermoScientific
1. First start day:
--> Ensure that the refrigerated trap contains a clean, dry, Glass Condensation Flask (GCF400) and that the supply of Thermo Scientific CryoCoolTM (SCC1) Heat Transfer Fluid is sufficient.
The CryoCoolTM in the refrigerated trap must be cold before drying runs can begin. Turn unit on at least 45 minutes before starting the drying run.

For best results, in our Core we maintain electrical power to the system (the main power switch on the front of the unit in the lower right is “ON” and the front display is illuminated) at all the times to keep the refrigerated trap cold and ready for use.
SECTION: Operating
2. Two modes of operation: with or without a fixed run time:
Manual run - Starts a “Manual” run based on the parameters loaded in the screen, use manual run function if you do not want to set a fixed run time.
Auto run - Starts an “Automated” run based on the parameters loaded in the screen, use auto run function if you want to set a fixed run time.
SECTION: Operating
3. Auto run for peptides in 70% ACN/0.1% F.A or 0.1% TFA:
The unit is turned on and the cover is unlocked.
The display is lighting up and shows the following in sequence:
Set values:
   Temperature 45°C
   Run time 2.00 h
   Vacuum Pressure 5.1 Torr 
   Ramp vacuum: 5
Open cover, add samples with the lids open and organized samples to be equilibrated.
Close cover
Click Auto-run
Pay attention to listen cover lock sound and air removal.
Wait few minutes and make sure the vacuum pressure is going down.
SECTION: Operating
4. Auto run for metabolites  in methanol/water:
The unit is turned on and the cover is unlocked.
The display lights up and shows the following in sequence:
Set values:
   Temperature 45°C
   Run time 4.00 h
   Vacuum Pressure 5.1 Torr 
    Ramp vacuum: 5
Open cover, add samples with the lids open and organized samples to be equilibrated.
Close cover
Click Auto-run
Pay attention to listen cover lock sound and air removal
Wait few minutes and make sure the vacuum pressure is going down.
SECTION: Operating
5. Auto-stop sound during auto run mode:
1 beep that is repeated until the user pushes the Stop button or opens the cover of the concentrator 
SECTION: Operating
6. Removing samples:
Click stop button
Open cover
Remove samples
Close cover
TITLE: ADI IsletCore Protocols for the Isolation, Assessment and Cryopreservation of Human Pancreatic Islets of Langerhans for Research Purposes
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This collection of protocols describe the processes used by the Alberta Diabetes Institute IsletCore for the isolation of human pancreatic islets of Langerhans for research. In addition, protocols related to quality control and the cryopreservation of human islets are included. </span><a href="http://www.bcell.org/adi-isletcore.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.bcell.org/adi-isletcore.html</span></a></div><div class = "text-block">The ADI IsletCore program is described in full here:</div><div class = "text-block"><span>Lyon J, Manning Fox JE, Spigelman AF, Kim R, Smith N, O'Gorman D, Kin T,  Shapiro AMJ, Rajotte RV, MacDonald PE (2016) Research-focused isolation  of human islets from donors with and without diabetes at the Alberta  Diabetes Institute IsletCore. </span><a href="http://press.endocrine.org/doi/abs/10.1210/en.2015-1562" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Endocrinology, 157(2): 560-569</span></a><span>.</span></div></div>

[STEPS]