eamag/protocols-with-steps huggingface dataset:
TITLE: Modified Zhen et al. SARS-CoV-2 Spike-Gene qRT-PCR assay for highly sensitive detection of the HV69/70 deletion
AUTHORS: d.noerz 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The SARS-CoV-2 B.1.1.7 lineage (British variant) features a number of hallmark mutations, which can be used for screening by conventional Taqman RT-PCR. A recently published highly sensitive diagnostic assay by Zhen et al. (J Mol Diagn, 2020) is coincidentally well suited to detect the HV69/70 deletion as its probe is located right on top of the mutation. In a sample with B.1.1.7 lineage, the assay returns a completely flat amplification curve, representing an assay drop-out phenomenon similar to what can be observed with the Thermo Fischer Taqpath test. We created a second Taqman prove (Probe-2) using locked nucleic acid technology to specifically target the same region including the deletion. In this way, the assay can be used (i.e. as part of a typing panel or multiplex) to detect the HV69/70-deletion in order to screen for different SARS-CoV-2 variants. </div></div>

[STEPS]
?. Modified Primer/Probe set based on Zhen et al., (J Mol Diagn, 2020): (5' - 3')S-gene fwdTCAACTCAGGACTTGTTCTTACS-gene revTGGTAGGACAGGGTTATCAAACS-gene P-1FAM-TGGTCCCAGAGACATGTATAGCAT-BHQ1S-gene P-2 (new)Yak-TGGTCCCAG(+A)(+G)AT(+A)GC(+A)T-BHQ1
S-gene fwdTCAACTCAGGACTTGTTCTTACS-gene revTGGTAGGACAGGGTTATCAAACS-gene P-1FAM-TGGTCCCAGAGACATGTATAGCAT-BHQ1S-gene P-2 (new)Yak-TGGTCCCAG(+A)(+G)AT(+A)GC(+A)T-BHQ1
?. Prepare a 4x primer/probe stock (500µL, for 100 reactions)S-gene fwd1004008S-gene rev1004008S-gene P-11001002S-gene P-21001002PCR-grade water--482
S-gene fwd1004008S-gene rev1004008S-gene P-11001002S-gene P-21001002PCR-grade water--482
?. Prepare RT-qPCR reaction-mix (100 reactions, final volume 1500µL). For this example, "RNA process control kit" (Roche) is used as one-step RT-PCR master.5x Roche one-step RNA process control master400200x Roche one-step RNA process control RT-Enzyme104x primer/probe stock500PCR-grade water590
5x Roche one-step RNA process control master400200x Roche one-step RNA process control RT-Enzyme104x primer/probe stock500PCR-grade water590
?. Add 5µL of sample RNA extract to 15µL of RT-qPCR reaction-mix per well in a 96-well PCR-plate. A z480 analyzer (Roche) was used to run the reaction according to the following protocol:RT50°C30 minutesDenature95°C15 minutesCycle - anneal60°C30 seconds|Cycle - denature95°C5 seconds45
RT50°C30 minutesDenature95°C15 minutesCycle - anneal60°C30 seconds|Cycle - denature95°C5 seconds45
?. Possible outcomes:FAM (Probe-1)strong signalnegativeYAK (Probe-2)weak signalstrong signalExemplary amplification curves and results of a clinical sample set using the protocol described above:Detection of the HV69/70 is not sufficient to confirm SARS-CoV-2 B.1.1.7 lineage in clinical samples. Screening for N501Y and P681H SNPs can be used to increase confidence prior to NGS.
FAM (Probe-1)strong signalnegativeYAK (Probe-2)weak signalstrong signal
TITLE: Blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using T4 DNA Polymerase.

[GUIDELINES]
CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´ → 5´ exonuclease activity of the enzyme. 

T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. Optimal activity is observed in NEBuffer 2.1. BSA supplementation is recommended when using a buffer that does not already contain BSA. 


References:

Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. In F. M. Ausebel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl(Ed.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

[STEPS]
1. Dissolve DNA in any 1 X supplemented with 100 Micromolar (µM) .
 

2. Add 1 unit of T4 DNA Polymerase per microgram DNA.
3. Incubate 15 min at 12 °C.
4. Stop reaction by adding EDTA to a final concentration of 10 Milimolar (mM).
 

5. Heat for 20 min at 75 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The six-minute walk test (6MWT) is a safe, simple, inexpensive tool for evaluating the functional exercise capacity of patients with chronic respiratory disease. However, there is a lack of standard reference equations for the six-minute walk distance (6MWD) in the healthy Chinese population aged 18-59 years.</p>

[BEFORE_START]
<p>The subjects spoke with the researcher to understand the study’s purpose and to complete a questionnaire to ensure that all subjects were healthy.</p>

[GUIDELINES]
<p>The six-minute walk test(6MWT) was completed according to the American Thoracic Society (ATS) guidelines.</p>

[STEPS]
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TITLE: AMOVA AND PHILOGENY IN SARS-COV- 2 PROTOCOLS
AUTHORS: Pierre Teodosio

[STEPS]
?. [METHODOLOGY]
1.METHODOLOGY
?. [METHODOLOGY]
DATABANK
?. [METHODOLOGY]
0pt;line-height:150%;font-family:"Arial",sans-serif;mso-ansi-language:EN-US'>: The 153 gene sequences of theintegrase gene of human immunodeficiency virus 1 were collected from GENBANK (https://www.ncbi.nlm.nih.gov/popset/?term=MN888087.1 and participate in a PopSet dipped by Totmenin and collaborators on March 25, 2020 (Popset:1822236350).
?. [METHODOLOGY]
PHYLOGENETICS ANALYSES: For phylogenetic analyses, the previously described nucleotide sequences were used. The sequences were aligned using the MEGA X program (TAMURA et al., 2018) and gaps were extracted for the construction of phylogenetic trees.
?. [METHODOLOGY]
GENETIC STRUCTURING ANALYSES: Paired FST estimators were obtained with the software Arlequin v. 3.5 (EXCOFFIER et al., 2005) using 1000 random permutations. The FST matrix generated by the software was used in the construction of a dendrogram based on the UPGMA distance method with the MEGA X software (TAMURA et al., 2018) and the FST and geographic distance matrices were not compared.
TITLE: The image processing protocol of “Comparison of an algorithm quantitatively estimating epifascial fibrosis in three-dimensional computed tomography images to other clinical lymphedema grading methods”
AUTHORS: Kyo-In Koo, Myung-hwan Ko, Yongkwan Lee, Son Hyewon, Suwon Lee, Chang Ho Hwang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The image processing protocol to recognize pixels suspected as reticulation in CT images. The processing sequences of the proposed algorithm submitted to PLOS ONE (eISSN: 1932-6203).</div></div>

[STEPS]
TITLE: Brain Dissection of Post-natal Mice
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for removal and embedding of the post-natal mouse brain.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Adapted from Steripak protocol, with addition of RNase.

[GUIDELINES]
Required volumes: for ½ plate 20 mls is plenty - split into 15 mls plus 5 mls; for a whole plate 35 mls is good - split into 30 mls plus 5 mls.

[STEPS]
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TITLE: Prodentim Reviews- Pills Price in Canada, UK, NZ, Australia or Ireland
AUTHORS: health  

[DESCRIPTION]
Don't buy before read Prodentim Reviews scam, price, review, results, side effects, benefits and much more.

[STEPS]
1. Prodentim Reviews there are plenty of alternatives you can do at the drugstore to get whiter teeth less than HR. See Compliance with Actions for even more information. Use the cost of baking salt. This is because baking soft drinks is easy to grungy that aids in removing stains from teeth. To start saturating your Mobile White Teeth Whitening amazon toothbrush you dip in some baking soda ingredients. Next, the composition of the comb teeth, as usual, paying special attention to 16 front teeth visibly. You need to comb with regard to three minutes. I remember that over time baking soda can wear down tooth enamel. , how you use, ingredients, composition, works (2) for this reason, it is not how you use ingredients a great idea to do this therapy every day. Do it once or twice a week to whiten your Prodentim Reviews teeth without hurting them. It uses hydrogen peroxide. You could use to get whiter teeth. It is completely risk free as long as you prevent swallowing. Soak the fabric order in the Hydrogen Peroxide Remedy. After that, use a damp cloth to gently massage your teeth. The hydrogen peroxide will get rid of the chemical stain, while the fabric will literally help remove it. After lunch, eat a row of strawberries as a party. is White, which helps tidy up and also dirties the teeth, making them whiter. Similarly, you can mash strawberries and mix them with baking soda to form a natural toothpaste. Other foods that help clean and whiten teeth are usually apples, pears, carrots. And celery. Especially avoid foods and drinks that can stain your teeth. Official Web. https://www.outlookindia.com/outlook-spotlight/prodetim-reviews-australia-nz-2022-scam-prodentim-uk-price-or-buy-in-prodentim-canada-or-united-states-news-207958
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol to prepare <em>E. coli</em> supercompetent cells to transform with plasmid/ligation. This protocol is originally derived from <a href="http://www.ncbi.nlm.nih.gov/pubmed/6345791" target="_blank">Hanahan, D. (1983) J. Mol. Biol. 166:557-580</a> with some changes. This version works exceptionally well for cloning PCR products where the number of colonies is not expected to be as high as from some ligations. These cells outperform commercially purchased supercompetent cells in my hands.

[BEFORE_START]
<strong>Prepare for day 2:</strong><br />
<ol>
<li>42˚C waterbath</li>
<li>37˚C waterbath</li>
<li>prechilled transformation tubes</li>
<li>37˚C SOC</li>
<li>LB amp+ Xgal agar plates</li>
</ol>
<h3><br /><strong>Buffers</strong></h3>
<h4><a target="_blank"></a><strong>TFB    Maniatis 1.78</strong></h4>
10mM K-MES (pH 6.2) adjust with KOH<br />100mM KCl<br />45mM MnCl2 <br />10mM CaCl2<br />3mM HACoCl3 (Haxamine cobalt III chloride)<br />filter sterilize and store at 4˚C (lasts a very long time)<br />
<h4><a target="_blank"><br /></a><strong>SOB</strong></h4>
2% bacto-tryptone (20gl<sup>-1</sup>)<br />0.5% yeast extract (5gl<sup>-1</sup>)<br />10mM NaCl<br />2.5mM KCl<br />10mM MgCl<sub>2</sub><br />10mM MgSO<sub>4</sub><br /><br /><strong>SOC</strong> is SOB with 20mM glucose

[STEPS]
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TITLE: Populating NCBI template for submissions using  BioNumerics  
AUTHORS: Ruth Timme, Maria Balkey, Julie Haendiges, Brian Sauders, Tina Lusk Pfefer

[DESCRIPTION]
PURPOSE: to define the standard operating procedure for collecting isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI.

SCOPE: to provide a standardized procedure to collect isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI.

RESPONSIBILITIES- SOP Responsible Officials: Ruth Timme, Maria Balkey

The GenomeTrakr Network Management will be responsible to monitor GenomeTrakr submissions processed through Bionumerics and ensure that all GT labs are familiar with the mandatory metadata fields required for submission of GenomeTrakr sequencing records to NCBI. 

V3:  Dropdown menus from controlled vocabulary added to the ncbi_update submission sheet

[STEPS]
SECTION: New Version for this protocol
1. Visit the new version of this protocol by clicking the following link: https://www.protocols.io/view/populating-ncbi-template-for-submissions-using-bio-3byl4qn4ovo5/v1
TITLE: PCL:PEG Electrospinning
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to electrospin a PCL:PEG copolymer onto glass cover slip.</div></div>

[STEPS]
?. [Make polymer solution]
In 50 mL tube, add chloroform and acetone. This is your solvent solution.
14 ml
6 ml
The solvent solution should be 70% chloroform and 30% acetone. You can adjust volumes accordingly.
Always work with these solvents in chemical fume hood.
?. [Make polymer solution]
In 15 mL tube dissolve  PCL in solvent solution to make a solution of PCL in solvent.
1 g
10 ml
10 Mass/Volume Percent
?. [Make polymer solution]
In a second 15 mL tube dissolve PEG in solvent solution to make a solution of PEG in solvent.
500 mg
10 ml
5 Mass/Volume Percent
?. [Make polymer solution]
Place the solvent solutions on a rocker overnight to fully dissolve.
?. [Load the dock]
Fold a 10 cm × 10 cm piece of aluminum foil.
?. [Load the dock]
Attach foil square to copper plate.
?. [Load the dock]
Using double-sided tape, attach cover slips onto aluminum foil.
?. [Load the dock]
Face the copper plate normal to the syringe pump within the enclosure, and move until the plate is approximately 9 cm away from the needle.
Increasing the distance will decrease fiber diameter.
?. [Load the dock]
Vortex the PCL:PEG solution for 3 minutes
?. [Make polymer solution]
Combine the polymer solutions at a rate of 3 parts PCL solution to 1 part PEG solution.
?. [Load the dock]
Pour PCL:PEG solution onto petri dish and draw solutin into a 10 mL syringe with 23 ga. tip.
?. [Load the dock]
Purge any bubbles from the syringe by holding the syringe with the tip facing upward and depressing the plunger.
?. [Load the dock]
Load syringe onto syringe pump, and secure.
?. [Electrospin]
Clip the ground (black) wire to the leads coming from the copper plate.Clip positive (red) wire to the syringe needle.
?. [Electrospin]
Set syringe pump rate to 20 µL/min, and begin pumping.
?. [Electrospin]
Turn on power source set to 15,000 V.
Do not touch any of the power source leads. Turn off power source to make any adjustments to the setup.
?. [Electrospin]
If necessary, "aim" the needle/syringe pump to obtain a uniform layer on all coverslips.
?. [Electrospin]
If clumping occurs at the needle, use a non-conducting material to clear.
?. [Harvest material ]
Once a desired thickness has been spun, turn off power supply and allow to cool for 2-3 min.
?. [Harvest material ]
Detach power supply leads.
?. [Harvest material ]
Use a blade to cut the fibers between the cover slips. Use caution not to disturb the fibers on the cover slips.
?. [Harvest material ]
Remove cover slips carefully.
TITLE: Automated 96-well PCR Purification
AUTHORS: Ariel Rabines, Rob Lampe, Andrew E Allen

[DESCRIPTION]
This protocol describes a fully-automated PCR cleanup in a 96-well PCR plate format with AMPure XP beads on an Eppendorf epMotion. Multichannel pipetting with the 300 uL and 50 uL pipettes is used. It is designed to use the thermomixer module; however, this can be replaced by modifying the program to mix by pipetting. The bead magnet used in this program is a Alpaqua Low Elution Magnet Plate. We also use Eppendorf twin.tec PCR plates (LoBind, skirted, 150uL).  

IMPORTANT: The program is currently set to add 22 µL of water to each well followed by 65 µL beads. This results in an approximately 1:1.5 bead ratio with a starting volume of 22 µL PCR product. Modify the first step accordingly with your starting volume and desired bead ratio.

The manufacturer's instructions are found here: https://www.beckmancoulter.com/wsrportal/techdocs?docname=B37419

The epMotion program is attached here.

[STEPS]
1. Take aliquot of beads out of refrigerator and allow to warm to room temperature.
4. Prepare fresh 70% EtOH. 

From the Agencourt XP beads manual: "Fresh 70% ethanol should be prepared for optimal results. There is also miscibility involved with ethanol and water. For example, measuring out 70 mL of ethanol and topping off to 100 mL with water will generate ~65% ethanol. Measuring 70 mL ethanol and 30 mL water separately, then combining them will generate ~95 mL of 70% ethanol."
2. Start program and input the numer of samples you have. It will then tell you the minimum volumes for each reagent. The positions in the reservoir rack are:
1. Molecular grade H2O
3. Beads
5. 70% EtOH
7. Elution buffer (e.g. H2O, TRIS-Acetate (10 mM pH 8.0), or TE Buffer (10 mM Tris-Acetate pH 8.0, 1 mM EDTA))

You can use the level sensor to detect volumes or measure them with a serological pipette. Do not start the program until all of your items are in the correct positions.
5. Place reagents, your PCR plate, and a new clean plate on the epMotion. 

Materials layout:
  


Run the program. It will take approximately 1 hour for a full plate.
3. Mix beads well until they appear homogenous.
TITLE: Consistency between anticholinergic burden scales in the elderly with fractures
AUTHORS: Luis Fernando Valladales Restrepo, Marlene Duran Lengua, Edgar Castro, Jorge Machado Alba

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Consistency between anticholinergic burden scales in the elderly with fractures</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Objective</span><span style = "font-style:italic;">: </span><span>falls and bone fractures are important causes of morbidity and mortality in the elderly. The objective of this study was to identify the degree of consistency between the anticholinergic scales used for patients diagnosed with fractures.</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Methods</span><span style = "font-style:italic;">: </span><span>this was an analytical agreement study conducted in patients diagnosed with vertebral and nonvertebral fractures in Colombia. The quadratic-weighted </span><span style = "font-style:italic;">kappa</span><span> coefficient was used to identify the consistency between the Anticholinergic Drug Scale-ADS, Anticholinergic Cognitive Burden Scale-ACB and Anticholinergic Risk Scale-ARS in assessing the prescriptions of fracture patients during the month prior to the fracture, during their stay as an inpatient and at discharge, according to Landis criteria.</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Results</span><span style = "font-style:italic;">: </span><span>220 patients with fractures were included, with a mean age of 75.3±10.3 years, and 68.2% were women. The ACB scale identified the highest anticholinergic burden (26.8%) in prescriptions made the month before the fracture, and the highest agreement was between ACB and ADS (0.717); during hospitalization and at discharge, the cholinergic antagonists were best identified with ADS (77.7% and 72.1%, respectively), with the best agreement between ACB and ARS (0.613 and 0.568, respectively). The prescription of tramadol was found in 64.1% of hospitalized patients and in 61.4% of patients at the time of discharge.</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Conclusions</span><span style = "font-style:italic;">: </span><span>the scales evaluated show marked discrepancies between them, with highly variable frequencies of anticholinergic drugs identified at the different prescription times, and with low agreement among them, which is why the scales are not interchangeable in patients with bone fractures.</span></div></div>

[STEPS]
TITLE: Immunocytochemistry (ICC)
AUTHORS: mineechoi

[DESCRIPTION]
This protocol describes how to do immunocytostochemistry for primary and hiPSC-derived cells. 

[STEPS]
SECTION: Cell fixation
1. Cells are fixed in 4 % volume paraformaldehyde (PFA)  and stored in phosphate-buffered saline (PBS) until use.





SECTION: Permeabilizing and blocking cells
2. Wash cells with PBS twice.
SECTION: Permeabilizing and blocking cells
3. Incubate the cells in 0.2 % volume Triton X-100, 5 % volume bovine serum albumin (BSA) for 60 min atRoom temperature. 
 
 


 



SECTION: Incubate cells in primary antibodies
4. 
 

Dilute primary antibody in 5 % volume BSA and incubate cells at 4 °C, 60 min or 60 min at Room temperature. 
 

SECTION: Incubate cells in primary antibodies
5. Wash cells with 5 % volume BSA for 5 min three times. 

SECTION: Incubate cells in secondary antibodies
7. Wash cells with 5 % volume BSA for 5 min three times away from light.
 



SECTION: Incubate cells in secondary antibodies
6. Dilute primary antibody in 5 % volume BSA and incubate cells at 4 °C 5 min or 60 minat Room temperature. 

SECTION: Incubate cells in secondary antibodies
8. Take away PBS and load anti-fading medium to cover cells.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from <span style="font-family: 'Segoe UI', sans-serif; font-size: small;">Schwyn, B. &amp; Neilands, J. B. Universal chemical assay for the detection and determination of siderophores.</span><span style="font-family: 'Segoe UI', sans-serif; font-size: small;"><em>Anal Biochem</em></span><span style="font-family: 'Segoe UI', sans-serif; font-size: small;">, 160:147-156 (1987).</span></p>

[STEPS]
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TITLE: BG11 hypersaline agar plates
AUTHORS: Bosak Lab Protocols

[STEPS]
?. Add the following to a beaker and dissolve using a stir bar on a magnetic stir plate.   ABC1BG11 hypersaline agar plate mediumamountunit2initial Milli-Q water968ml3NaCl49.8g 4NaNO31.5g5Na2CO30.02g6KCl1.3867g7MgCl2*6H2O8.569g8MgSO4*7H2O6.4998g9CaCl2*2H2O2.042g10Stock A10ml11Stock B10ml12Stock C10ml13Stock 5 (trace
  metals)1ml14Vitamins1ml15Agar15g
ABC1BG11 hypersaline agar plate mediumamountunit2initial Milli-Q water968ml3NaCl49.8g 4NaNO31.5g5Na2CO30.02g6KCl1.3867g7MgCl2*6H2O8.569g8MgSO4*7H2O6.4998g9CaCl2*2H2O2.042g10Stock A10ml11Stock B10ml12Stock C10ml13Stock 5 (trace
  metals)1ml14Vitamins1ml15Agar15g
?. Add Milli-Q water to bring total volume to
1 L
?. Autoclave media
?. While waiting for autoclave cycle to finish, set up desired number of sterile petri dishes or other culturing plates in a sterile biohood.
?. Remove media from autoclave and let cool until just warm enough to touch.
?. Pour media into each petri dish and let solidify.**TIP: Before agar solidifies, use a sterile pipette tip to move any bubbles that may form to the edges of the petri dish.
TITLE: Chiral resolution process
AUTHORS: BOC      Sciences

[DESCRIPTION]
The separation of a racemic mixture into two optically active forms (+ or −) is known as chiral resolution. Since diastereomers have different chemical and physical properties, they can be separated into corresponding enantiomers by chiral resolution. This method is called enantiomeric enrichment, and it is a process of continuously increasing the percentage of enantiomers until the enantiomeric excess finally approaches 100%.

[STEPS]