eamag/protocols-with-steps huggingface dataset:
TITLE: General Assembly and Alignment in Geneious
AUTHORS: Andrew J. Johnson, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to conduct general assembly and alignment of sequences in Geneious.</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>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Library building protocol for archival samples with single 8-nt adapters for Illumina platforms.</p>

[BEFORE_START]
<p>Calculate the DNA input and prepare the library adapters.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for plasmid minipreps that does not involve any columns and uses commonly accessible reagents. <br />While it doesn't result in highly pure DNA, it can provide a sufficient quantity of plasmid for sequencing, restriction analysis, preparative digests.</p>
<p>Beware of the large amount of RNA that co-purifies with plasmid DNA (it acts as an efficient carrier during the nucleic acid precipitation step). This contamination will make it impossible to determine DNA concentration from standard 280-nm absorbance readings.<br /><br /><strong>Credits:</strong> I first learned this protocol from the old-style protocols for bacmid purification using the Bak-to-Bac system (Invitrogen). <br />In that system, the large (&gt;50 kb) bacmid molecules were deemed too fragile for the Qiagen spin columns.</p>

[GUIDELINES]
<h1><strong>Qiagen plasmid purification buffers (from <a href="http://openwetware.org/wiki/Qiagen_Buffers" target="_blank">OpenWetWare.org</a>)</strong></h1>
<h4> </h4>
<h4><strong>Buffer P1</strong></h4>
<p><span style="font-weight: 400;">    * 50 mM Tris-HCl pH 8.0</span></p>
<p><span style="font-weight: 400;">    * 10 mM EDTA</span></p>
<p><span style="font-weight: 400;">    * 100 ug/ml RNaseA </span></p>
<p> </p>
<p><span style="font-weight: 400;">The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).</span></p>
<p><span style="font-weight: 400;">Note: LyseBlue reagent (1000X = 43 mg/ml thymolphthalein in ethanol)</span></p>
<h4> </h4>
<h4><strong>Buffer P2</strong></h4>
<p><span style="font-weight: 400;">    * 200 mM NaOH</span></p>
<p><span style="font-weight: 400;">    * 1% SDS </span></p>
<p> </p>
<h4> </h4>
<h4><strong>Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits; N3 contains the equilibration buffer, while P3 does not)</strong></h4>
<p><span style="font-weight: 400;">    * 3.0 M potassium acetate pH 5.5 </span></p>

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Purpose</strong>: To concentrate harvested phage lysate up to 20X - 40X. The Vivaflow device uses a peristaltic pump setup to recirculate the input sample over a 100,000 MWCO PES membrane, which allows the media filtrate to be diverted and the phage to remain in the starting sample.</p>

[BEFORE_START]
<p><strong><em>MODIFICATION NOTES: </em></strong></p>
<p> </p>
<p><em>To ensure full phage recovery from large sample starting volumes of up to 4-8 L of lysate, some steps were added to the manufacturer’s instructions based on empirical trials of filtering capacity. The first trial was done following the Sartorius user manual steps explicitly for 8 L filtered on a Vivaflow 200 device, but resulted in about 25% phage recovery. The second trial incorporated modifications which enhanced recovery to nearly 100% from 4 L of lysate using a Vivaflow 200 device and 2 L of lysate using a Vivaflow 50 device, and is described in this protocol for the Vivaflow 50. This may not be an issue for sample volumes of less than 2 L being concentrated on the same device (Vivaflow 50), but this was not verified. </em></p>
<p> </p>
<p><em>Filtration of 8 L was successful using two of the Vivaflow 50 cassettes in parallel, taking about the same amount of time as using one Vivaflow 200 cassette (~1 L per hour); since the Vivaflow 50 set of 2 cassettes are much less expensive, though not reusable, we chose to use these to avoid any potential cross contamination of samples from repeated use (reuse is specified for the 200 and 50R devices).</em></p>
<p> </p>
<p><strong><em>Materials Notes:</em></strong></p>
<ul>
<li> <em> To use the Masterflex tubing and pump head (size 25) specified below with the Vivaflow 50 PVC tubing (size 16) provided, </em><em>modify the larger diameter (size 25) tubing with an appropriate adapter fitting. Attach the adapter fitting to the Masterflex tubing first and autoclave sterilize this together in a foil wrap. When ready to use, connect the larger and smaller tubing via the adapter, using sterile technique.</em></li>
</ul>

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TITLE: Nuclear cytoplasmic fractionation
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes nuclear cytoplasmic fractionation.

[STEPS]
SECTION: Nuclear cytoplasmic fractionation
1. Treat 500,000 BMDM’s with DMSO or 50 nanomolar (nM) MLi-2 for 360 min.
SECTION: Nuclear cytoplasmic fractionation
2. After the treatment, wash the cells 3X in PBS.
SECTION: Nuclear cytoplasmic fractionation
3. Harvest the cells in 500 µL of ice-cold hypotonic buffer.
SECTION: Nuclear cytoplasmic fractionation
4. Homogenize the cells with 20 strokes of a Dounce homogenizer.
SECTION: Nuclear cytoplasmic fractionation
5. To 100 µL of this homogenate, add SDS (1% final) and 25 U of Benzonase (Novagen). Use this as total lysate.
SECTION: Nuclear cytoplasmic fractionation
6. Spin the rest of the homogenate at 14000 rpm, 5 min, 4 °C.

SECTION: Nuclear cytoplasmic fractionation
7. Collect the supernatant (cytoplasmic fraction) into a new tube.
SECTION: Nuclear cytoplasmic fractionation
8. Resuspend the pellet (nuclear fraction) in 200 µL of high-salt buffer and solubilize with SDS (1% final) in the presence of 25 U of Benzonase.
SECTION: Nuclear cytoplasmic fractionation
9. Measure protein concentration with the BCA reagent (Thermo Scientific), and subsequently analyze the samples by immunoblotting.
TITLE: Xp Keto Gummies
AUTHORS: Pure  Keto

[DESCRIPTION]
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[STEPS]
TITLE: Brimblecombe, K.R. et al. (2023) Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine, co-varies with risk factors for Parkinson’s. 
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
This collection contains three protocols detailing methods used in Brimblecombe, K.R. et al. (2023) Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine, co-varies with risk factors for Parkinson’s. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol corrects small RNAs deep sequencing biases caused by RNA ligases. The key modification is the use of pooled adapters with the sequences below. See the cited publication for further details.

[BEFORE_START]
5' adapter-4N: 5’GUUCAGAGUUCUACAGUCCGACGAUCNNNN
<br>3' adapter-4N: 5’ rApp /NNNNTGGAATTCTCGGGTGCCAAGG/ 3ddC/
<br>Reverse primer: ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGXXXXXXTAGAGCATACGGCAGAAGACGAAC
<br>Forward primer: AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA

[STEPS]
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TITLE: One enzyme reverse transcription qPCR using Taq DNA polymerase
AUTHORS: Sanchita Bhadra, Andre Maranhao, Andrew Ellington

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.</div></div>

[STEPS]
?. [Preparing RT-qPCR reaction]
RT-qPCR assays were assembled in a total volume of   containing the indicated buffer at 1X strength (see Generation 6A buffer recipe in step 2)
25 µl
?. [Preparing RT-qPCR reaction]
The buffer was supplemented with  ,  , , and 2.5 units of Taq DNA polymerase from indicated commercial vendors.
[deoxyribonucleotides (dNTP)]
[each of forward and reverse PCR primer pairs]
[of the TaqMan probe]
?. [Preparing RT-qPCR reaction]
Indicated copies of SARS-CoV-2 viral genomic RNA, SARS-CoV-2 N gene armored RNA, or RNaseP armored RNA prepared in TE buffer (, , , ) immediately prior to use were added to RT-qPCR reactions containing corresponding PCR primers. Negative control reactions did not receive any specific templates.
[Tris-HCl]
[EDTA]
?. [Running RT-qPCR]
Amplicon accumulation was measured in real-time by incubating the reactions in a LightCycler96 qPCR machine (Roche, Basel, Switzerland) programmed to hold    for  followed by  for  prior to undergoing 55 cycles of  for  and  for . TaqMan probe fluorescence was measured during the amplification step (60 °C for 30 sec) of each cycle using the FAM channel.
60 °C
95 °C
95 °C
60 °C
?. [Optimized buffer recipe]
In this study, we report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions.We recommend to use Generation 6A buffer for the one enzyme RT-qPCR. 10X Generation 6A buffer (,,  ,  , )
[Tris-HCl]
[ (NH₄)₂SO₄]
[KCl]
[MgCl2]
TITLE: DNA extraction from mouthwash samples using Qiagen DNeasy PowersoilPro Kit
AUTHORS: Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Mamon Abd AlBaqi, Aashish R Jha

[DESCRIPTION]
This protocol describes how to extract DNA from mouthwash samples. 

[BEFORE_START]
Set centrifuge to 4ºC
Keep CD2 solution on ice or at 4ºC
Prepare and label collection tubes, microcentrifuge tubes, MB spin columns, and powerbead tubes 



[STEPS]
1. Thaw the sample on ice for 30 min 


2. Centrifuge the transferred sample at maximum speed (17000 x g ) at 4°C for 10 min 

1.1. Transfer the desired sample volume 1-2 mL  into a 1.5mL or 2mL eppendorf tube

3. Discard the supernatant carefully without disturbing the pellet 
3.1. Repeat Step 1.1 if more volume is needed to see a pellet or a higher yield of DNA is required
4. Add 800 µL of CD1 and vortex to resuspend pellet

4.1. Spin down briefly
5. Transfer entire eppendorf content in a PowerBead Pro Tube
6. Secure PowerBead Pro tube onto the bead beater and run at maximum speed (17000 x g ) for 5 min 

7. Centrifuge the PowerBead Pro tube at maximum speed (17000 x g ) for 90 s 

8. Transfer the supernatant carefully, without disturbing the pellet, into a clean 2mL microcentrifuge tube 
9. Add 200 µL of CD2 into the 2mL microcentrifuge tube and vortex for 10 s 

10. Centrifuge the 2mL microcentrifuge tube at maximum speed (17000 x g ) for 90 s 

11. Transfer the supernatant carefully without disturbing the pellet, into a clean 2mL microcentrifuge tube 
12. Add 600 µL of CD3 into the 2 mL microcentrifuge tube and vortex for 10 s 

13. Load 650 µL of the lysate onto MB spin columns and centrifuge at maximum speed (17000 x g ) for 90 s 

13.1. Discard flow-through and repeat step 13 to consume all the lysate 
14. Place the spin column onto a new collection tube, add 500 µL of EA, and centrifuge at maximum speed (17000 x g ) for 90 s 

15. Discard flow-through and place the spin column back into the collection tube 
16. Add 500 µL of C5 onto the spin column and centrifuge at maximum speed (17000 x g ) for 90 s 

17. Discard flow-through and place the spin column back into the collection tube 
18. Centrifuge at maximum speed (17000 x g ) for 2 min and place spin column into 1.5mL eppendorf (elution) tube 

19. Add 50-100 µL of nuclease-free water to the center of the spin column and leave at room temperature for  5 min 

20. Centrifuge at maximum speed (17000 x g ) for 1 min , quantify using Qubit, flash freeze, and store at -20/80 °C
TITLE: The Nanopore libraries preparation for the Scapharca broughtonii
AUTHORS: Chang-Ming Bai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to outline the process of Nanopore library preparation for </span><span style = "font-style:italic;">Scapharca broughtonii </span><span>genome.</span></div></div>

[STEPS]
?. About 6 μg genomic DNA was fragmented using a Megaruptor (Diagenode, Seraing, Belgium) to obtain ~20kb frequments, and verified with Agilent Bioanalyzer 2100 system  (Agilent Technologies, Palo Alto, CA, USA).
The sizes of the main fragments should >17 Kb.
?. Fragments with size > 15 Kb were selected using BluePippin Size-Selection system  (Sage Science, Beverly, the U.S.), and further purified using AMPure XP beads (Agencourt, Beverly, the U.S.).
?. DNA fragments were end-repaired using NEBNext FFPE Repair Mix (NEB, M6630). The precedures was keeped at 20℃, 15 min.
?. The repaired fragments were purified using AMPure XP beads (Agencourt, Beverly, the U.S.).
?. DNA fragments were end-repaired and dA-tailed by using the NEBNext End repair / dA-tailing Module (NEB, E7546). The precedures was keeped at 20℃, 30 min, and then at 60℃, 30 min.
?. The repaired fragments were purified using AMPure XP beads (Agencourt, Beverly, the U.S.).
?. Nanopore adaptors were ligated to the dA-tailed DNA fragment by using Ligation Sequencing Kit 1D R9 Version (Nanopore, SQK-LSK108) and NEB Blunt/TA Ligase Master Mix (NEB, M0367). The precedures was keeped at 20℃, 60 min.
?. The concerntration of the library was detected with Qubit 3.0 (Thermo Fisher Scientific Inc., Carlsbad, CA, USA), and proved to be qualified for sequencing.
?. This was carried out with Ligation Loading Bead Kit R9 Version (EXP-LLB001) and MinION Flow Cell (FLO-MIN106) according to the user manual.
?.
TITLE: Detection of Human Rhinovirus by PCR following Sanger Sequencing
AUTHORS: Talita Gagliardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Reference for the method and primer sequences</div><div class = "text-block">“Lee W.-M., Lemanske R. F., Evans M., Vang F., Pappas T. E., Gangnon R., Jackson D. and Gern J. E.. 2012. Human Rhinovirus Species and Season of Infection Determine Illness Severity. American Journal of Respiratory and Critical Care Medicine. Submitted.”
</div><div class = "text-block">“Lee W.-M., Kiesner C., Pappas T., Lee I., Grindle K., Jartti T., Jakiela B., Lemanske R.F., Shult P.A., Gern J.E. 2007. A diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants. PLoS ONE 2:e966.”

</div></div>

[STEPS]
?. Prepare the PCR reaction using 3ul of random cDNA; 10pM of the mixture of forward primers (B1, B2 and B3) and 10pM of the FR2 reverse primer.
?. Run a Touch-down PCR following the cycling conditions suggested by the polymerase manufacturer although varying the annealing temperature from 68C to 52C (decreasing 2C after 2 cycles).
?. Purify 5ul of the product from the first PCR using “ExoSAP-IT Express PCR Cleanup Reagent” (Thermo) and manufacturer’s protocol.
?. Prepare again the PCR reaction using 3ul of purified amplicon; 10pM of the mixture of forward primers (B1, B2 and B3) and 10pM of the FR2 reverse primer.
?. The PCR cycling conditions should be set as suggested by the polymerase manufacturer, but this time the  annealing temperature keeps set as 52C.
?. Purify 10ul of the product from the second PCR using ExoSAP-IT and manufacturer’s protocol.
?. Use this purified amplicon in a Sanger sequencing with the FR2 reverse primer only.
?. Analyze the chromatogram and use the nucleotide sequence to search in a database as BLAST.
TITLE: Keto Blast Gummies Shark Tank Reviews- Price or Ingredients
AUTHORS: health  

[DESCRIPTION]
Keto Blast Gummies Reviews -Many users are convinced of the effectiveness of the Keto Blast Gummies. You are particularly enthusiastic about the ease of use and effect. Some buyers report that they have tried many diets in the past. This prevented them from reaching their dream weight.

[STEPS]
1. Keto Blast Gummies Reviews -Many users are convinced of the effectiveness of the Keto Blast Gummies. You are particularly enthusiastic about the ease of use and effect. Some buyers report that they have tried many diets in the past. This prevented them from reaching their dream weight. Keto Blast Gummies is definitely an all-natural health supplement which can be created for people who have already been using a ketogenic diet program, staying away from most carbohydrates. The product accentuates the results of the diet regime, improving the user to quickly burn off fat and shed extra pounds. The nutritional supplement offers the customer with a lot more energy levels. This manufacturer offers diet nutritional supplements that concentrate especially on the well known keto eating plan, increasing the weight losses effect. These vitamin supplements use only organic and natural, 100 % natural ingredients without any synthetic or substance compounds. This supplementation is meant for individuals who already are after a ketogenic diet plan. What this means is they are limiting carbs and eating many proteins and fats. When you repeat this, your system gets deprived for sugar. The liver organ then releases ketone body in your method, and so they assist you to start out burning fat as electricity in lieu of sugar. Keto Blast Gummies works  effectively in giving you toned shaped body without giving you any side  effects. This formula speeds up the process of ketosis in your body  which helps in melting all the excess body weight and converted into  higher energy levels. This formula reduces your belly fat and boosts  your digestion and immunity power so that you can overcome any health  issues safely without having any harsh impact. It helps in  controlling your hunger and cravings so that you don’t gain excess body  weight. Official Web: https://www.outlookindia.com/outlook-spotlight/keto-blast-gummies-canada-reviews-shark-tank-keto-blast-gummy-bears-canada-price-where-to-buy-shocking-scam-exposed-2022--news-210938
TITLE: LRRK2 microtubule sedimentation binding assay
AUTHORS: Andrea Dickey, Mariusz  Matyszewski

[DESCRIPTION]
Assay to determine LRRK2 protein binding to microtubules.

Original assay by Andrea Dickey. Adapted by Mariusz Matyszewski for protocols.io.

Assay originally used in Snead, Matyszewski, Dickey et al. 2022

[STEPS]
SECTION: Microtubule preparation
1. Polymerize tubulin at around 2.5 mg/mL  for 30 min at 37 °C . Add Taxol for stabilization and incubate for another 10 min at 37 °C .

SECTION: Microtubule preparation
2. Remove free tubulin by ultracentrifugation. 108628 x g, 15 min, 37 °C through a 64 % volume .

SECTION: Microtubule preparation
3. Resuspend the resulting microtubule pellet in the LRRK2 binding buffer.
SECTION: Microtubule preparation
3.1. Determine microtubule concentration by running an SDS-PAGE with actin standards.
SECTION: Microtubule preparation
4. Incubate desired amount of LRRK2RCKW protein (200 nanomolar (nM) in our experiments) at Room temperature for 10 min with varied concentrations of microtubules in the LRRK2 binding buffer.


 

SECTION: Microtubule preparation
5. Pellet the microtubules by ultracentrifugation. 108628 x g, 15 min, 25 °C 

SECTION: Microtubule preparation
6. Quantify the depletion of LRRK2RCKW by taking the supernatant and boiling for 10 min in SDS containing buffer for running a gel.

SECTION: Microtubule preparation
7. Samples were run on 4-12% polyacrylamide gels (NuPage, Invitrogen) and stained with SYPRO-Red Protein Gel Stain (ThermoFisher) for protein detection.
Binding curves were fit in GraphPad Prism (9.2; GraphPad Software) with a nonlinear regression hyperbolic curve.
TITLE: Determination of Effective Synaptic Conductances Using Somatic Voltage Clamp
AUTHORS: Songting Li, Nan Liu, Xiaohui Zhang, Douglas Zhou, David Cai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">By using dynamic clamp to generate synaptic inputs, we demonstrate the deficiency of the traditional slope-and-intercept method for the determination of effective synaptic conductances using somatic voltage clamp, and  and we test the effectiveness of the intercept method for the measurement of effective conductance.</div></div>

[STEPS]
?. Clamp the somatic voltage at various levels using voltage clamp, generate simultaneous E and I synaptic input currents using dynamic clamp on the dendrite, and measure the corresponding synaptic currents arriving at the soma. In this step, one shall exert a well control of the experimental condition such that the E and I synaptic inputs received by the neuron under different holding voltage are approximately the same.
?. Fit a linear relation between the holding voltage and the synaptic current and read out the intercept value from the I-V relation at each time point. Holding potentials of recorded cells shall be corrected for a calculated liquid junction potential (Barry etal, Journal of Neuroscience Methods, 1994).
?. Vary the reversal potential to a different value through dynamic clamp and repeat the first two steps to obtain its corresponding intercept value at each time point.
?. Recover the effective E and I condcutances through the intercepts of the two I-V relations.