eamag/protocols-with-steps huggingface dataset:
TITLE: Chimeric Protein-LAG and Staphylococcal Protein A sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein-LAG (PLAG) and staphylococcal protein-A (SpA) with different immunoglobulin preparations of mammalian and avian species. </div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein-LAG (PLAG) and staphylococcal protein-A (SpA) with different immunoglobulin preparations of mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpA conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.30.
TITLE: Clinical education alone is sufficient to improve strength training exercise prescription
AUTHORS: A/Prof Gavin Williams, Prof Linda Denehy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The main cause of reduced ability to walk for the majority of people with neurological conditions is muscle weakness. Clinical guidelines from the National Stroke Foundation (NSF) Clinical Guidelines for Stroke Management and the American Heart Association Statement for Stroke both recommend strength training for the lower limb to improve walking. Further, clinical guidelines have also been published by the American College of Sports Medicine (ACSM) for optimal strength training. However, despite a large body of evidence that muscle weakness is the primary impairment causing walking limitations, and guidelines recommending strength training during rehabilitation, six systematic reviews have demonstrated that the application of the NSF guidelines has not improved walking outcomes. Our research indicates that the gap is within the NSF guidelines. The NSF guidelines are vague and lack the ‘How To’ direction for the implementation of their strength training recommendations. The aim of this research project is to develop an education and training package for implementing the ACSM guidelines for strength training, and test whether it is effective in order to direct the application of the NSF guidelines.</div></div>

[STEPS]
TITLE: Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes general procedures used for subcutaneous injection in adult mice.</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]
<div title="Page 1">
<div>
<div>
<p>Endo F3 cleaves free or Asparagine-linked triantennary or biantennary oligosaccharides,as well as triamnnosyl chitobiose core structures. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamineresidue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Alpha 1-3 fucosylation will inhibit enzymatc activity.</p>
</div>
</div>
</div>

[STEPS]
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TITLE: Do, Q. B. et al. (2023) Early striatal hyperexcitability in an in vitro human striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation
AUTHORS: Quyen Do, Bryan Ng, Ricardo Marquez Gomez, Dayne Beccano-Kelly, Naroa Ibarra-Aizpura, Maria-Claudia Caiazza, Charmaine Lang, Jimena Baleriola, Nora Bengoa-Vergniory, Richard Wade-Martins

[DESCRIPTION]
This collection contains eight protocols detailing methods used in Do, Q. B. et al. (2023) Early striatal hyperexcitability in an in vitro human striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Optimised protocol for the isolation of non-parenchymal cells from human liver biopsies. Used on 1-2cm3 biopsies. 

[GUIDELINES]
Process tissue ASAP after removal from patient to avoid excessive cell death.

[STEPS]
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
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SECTION: Dissociation
?. 
SECTION: FACS for Single Cell Analysis
?. 
SECTION: FACS for Single Cell Analysis
?. 
SECTION: FACS for Single Cell Analysis
?. 
SECTION: FACS for Single Cell Analysis
?. 
SECTION: FACS for Single Cell Analysis
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Electroporation strategy used in the transformation of Aurantiochytrium limacinum (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid). This protocol is following the guidance of Ono et al. 2011. US Patent # 7,888,123 B2. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Host Decoy Traps (HDT) sample outdoor-biting mosquitoes and have potential as a monitoring malaria vectors. The trap emits human odour, is visually conspicuous and heated to human body temperature, thus mimicking a human host, and therefore, it catches a proportion of the number of mosquitoes that would land on a host with the same characteristics. For example, at low population densities of <em>Anopheles coluzzii</em>, the HDT can catch 1.5x that of a standard human landing catch, and in the rainy season this increased dramatically; the HDT can catch 10x the number in a human landing catch.</p>
<p> </p>
<p>The trap consists of an adhesive trapping unit and an odour source. Human odour from a volunteer in a tent is blown down the plastic pipe and around the warm visually conspicuous trap. Mosquitoes are attracted to the odour. The high visual contrast of the black trap against the soil is seen by mosquitoes and they fly close to the trap to determine if it is the host. At close range, mosquitoes detect the relative warmth of the trap compared to the ambient air temperature and this induces landing in search of a suspected host, causing the mosquito to become stuck on the adhesive plastic surface.</p>

[STEPS]
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TITLE: Protocols for Molecular Characterization of the Female Reproductive System
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
Here we describe our multi-step protocol to generate a comprehensive molecular characterization of the female reproductive system. We begin with organ procurement that maintains functional characteristics of the uterus, ovaries, and Fallopian tubes and then describe our structured tissue sampling procedure that represents anatomical, physiological, and individual diversity of the female reproductive system, toward full exploration of the function and structure of female reproductive cells. 

These protocols were developed as part of the National Institutes of Health (NIH) Human BioMolecular Atlas Program (HuBMAP), we are centered around the 10X Genomics Multiome and 10X Genomics Visium platforms.


[STEPS]
1.   

2. Preparation and Preservation of the Female Reproductive System (ovaries, Fallopian tubes and uterus) Prior to Procurement
https://dx.doi.org/10.17504/protocols.io.ewov1nr57gr2/v1
3. Removal of the Female Reproductive SystemEn Bloc
https://dx.doi.org/10.17504/protocols.io.bp2l61jrzvqe/v1
4. Post-Surgical Dissection of Ovaries
https://dx.doi.org/10.17504/protocols.io.j8nlkkzn1l5r/v1
5. Post-Surgical Dissection of Fallopian Tubes
https://dx.doi.org/10.17504/protocols.io.14egn75eqv5d/v1
6. Post-Surgical Dissection of Cervix
https://dx.doi.org/10.17504/protocols.io.5jyl89467v2w/v1
7. Post-Surgical Dissection of Uterine Body
https://dx.doi.org/10.17504/protocols.io.8epv59yb4g1b/v1
8. Tissue Slice Preparation for Visium Analysis
https://dx.doi.org/10.17504/protocols.io.eq2lyno9qvx9/v1
9. Manual Tissue Dissociation for Multiome Analysis
https://dx.doi.org/10.17504/protocols.io.8epv59y34g1b/v1
10. Tissue Dissociation for Multiome Analysis Using S2 Singulator
https://dx.doi.org/10.17504/protocols.io.yxmvmndx6g3p/v1
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Reagent List:</strong></p>
<p> </p>
<ul>
<li>Sterile PBS</li>
<li>Cell culture medium (RPMI 1640 supplemented with 10% FBS)</li>
<li>Sterile 12-well plate</li>
<li>Sterile 6-well plate</li>
<li>RBC Lysis Buffer (Cat. No. 420301)</li>
<li>Anti-mouse CD3ε, clone 145-2C11 (LEAF™ format, Cat. No. 100314)</li>
<li>Anti-mouse CD28, clone 37.51, (LEAF™ format, Cat. No. 102112)</li>
<li>Anti-mouse IL-4, clone 11B11, (LEAF™ format, Cat. No. 504108)</li>
<li>Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)</li>
<li>Recombinant mouse IL-12 (p70) (carrier-free) (Cat. No. 577002)</li>
<li>Monensin Solution (Cat. No. 420701)</li>
<li>PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)</li>
<li>Ionomycin (Cat. No. I0634 from Sigma)</li>
</ul>

[STEPS]
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TITLE: Microscope video recording
AUTHORS: Daniel  Vaulot

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to record videos on SBR Microscope</div></div>

[STEPS]
?. [First steps]
Prepare your sample
- Cultures flask
- Drop between slide and coverslip and invert the slide so that the objective is on side of coverslipUse x 40 objective for most cultures and x20 for dinoflagellatesInstall and start Infinitity Capture software
?. [Acquisition with Infinity Capture]
Infinity X - 800x608 (4:3) resolution for color videos - Note that only part of the field is visible but files are smaller than with full resolutionInfinity 3-6 UR 1024x768 ou 1600x1200
?. [Acquisition with Infinity Capture]
Use 960x728 (4:3) resolution (binning) for BW  videos - Note that the full field is visible
?. [Acquisition with Infinity Capture]
Modify the AVI setupsLaunch acquisition (usually only 10-20 sec at a time)
?. [Acquisition with Infinity Capture]
Infinity X - Full resolution 1920x1080 (16:9) - Note : very large AVI files ! 30 sec = 700 MoInfinity 3UR-6 - Full resolution 2752 x 2192
?. [Acquisition with Infinity Capture]
Advantage : Very easy to doDisadvantage : Files are store as avi and are very very large...
?. [Acquisition with Video de Luxe]
Need to buy the software (40 euros only)Advantage : Much smaller files and the software allow to finalize the films
?. [Acquisition with Video de Luxe]
Setup the resolution in Image CaptureShut donw Image CaptureLaunch Video de LuxeSelect in menu : Video Recording (Enregistrement video)Slect the USB Camera - DShow (see next step)
?. [Acquisition with Video de Luxe]
?. [Acquisition with Video de Luxe]
?. [Make the final movie with Video de Luxe]
Create a new movieSelect the parts you want to addCut the scenesOptimum duration is 20-30 sec max
?. [Make the final movie with Video de Luxe]
?. [Make the final movie with Video de Luxe]
Export to wmv format (Windows Movie - this is the most compact format without loss of resolution)Choose a format with same aspect ratio than originalDo not add sound unless you want to add musicFor 16:9 : 640 x 360 or 1280 x 720For 4:3 : 640 x480
?. [Upload to YouTube]
?. [Acquisition with Video de Luxe]
Set up configuration as follows (item 3 in previous step)Infinity X - Resolution 1280 x720Infinity 3-6UR - Resolution 1024x768 (4:3)Ratio 16:9
TITLE: Removal of Single-Stranded Extension using Mung Bean Nuclease (M0250)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the protocol for the removal of single-stranded extension using Mung Bean Nuclease (M0250). 

[STEPS]
1. Suspend DNA (0.1 μg/μl) in 1X Mung Bean Nuclease Buffer or 1X NEBuffer 1.1, 2.1 or CutSmart®.
2. Add 1.0 unit of Mung Bean Nuclease per μg DNA.  
3. Incubate at 30 °C for 30 min. 
4. Inactivate the enzyme by phenol/chloroform extraction or by addition of SDS to 0.01%.
5. Recover the DNA by ethanol precipitation.
TITLE: Power in a single case multiple baseline design
AUTHORS: Samantha Bouwmeester, Joran Jongerling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block">A randomization test can be used to statistically test hypotheses in multiple baseline designs to complement the commonly used visual inspection analysis. A crossed factor simulation study was performed to investigate the power of the Koehler and Levin (1998)randomization test in an multiple baseline design. The results show that the degree of autocorrelation of the observations, the number of participants, the effect size, the overlap of possible start moments of the intervention between participants, the ratio of the number of measurements in the baseline- and intervention phase, a gradually emerging effect, and the number of measurements had strong main effects on the power. The two-way interactions between number of participants and effect size, and between the number of measurements and the number of start moments of the intervention also had a large effect. An online tool was developed to calculate the power of a multiple baseline design given several design characteristics.</div></div>

[STEPS]
TITLE: Variant (E484K) ALERT - Ligation-Dependent Loop-Mediated Isothermal Amplification
AUTHORS: Ali Bektas, Anitha Jayaprakash

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A 2-hour, 2-temperature protocol, using RNA templated DNA ligation, for the visual detection of the E484K mutation of concern pertaining to SARS-CoV-2.</div><br/><div class = "text-block">N.B. This is the first version of this protocol, stay tuned for increased sensitivity and multiplexing (A.B.)</div></div>

[STEPS]
?. [Ligation ]
X1 Ligation mix (multiply for number of samples processed) E484K-Donor (100nM) E484K-Acceptor (100nM)SplintR Buffer (NEB) (NEB) molecular biology grade wateradd  RNA per  of reaction mix for a  reaction volume
2 µl
2 µl
1 µl
0.2 µl
1.8 µl
3 µl
7 µl
10 µl
?. [Ligation ]
Incubate at  for  followed by a  at  inactivation step.
37 °C
65 °C
?. [Ligation ]
Place ligation reactions on ice.
?. [LAMP ]
X1 LAMP mix (multiply for number of samples processed) Isothermal Buffer 1 (NEB) (100mM) dNTPs (10µM) FIP (50µM) BIP (50µM) F3 (10µM) B3 (10µM) (100µM) Molecular Biology Grade wateradd  of the ligation product from the Ligation step to  of LAMP mix for a  reaction.
2.5 µl
0.5 µl
3.5 µl
0.8 µl
0.8 µl
0.5 µl
0.5 µl
0.1 µl
1 µl
11.8 µl
3 µl
22 µl
25 µl
?. [Ligation Primers]
Oligonucleotides used for RNA templated DNA ligation using All sequences shown 5' to 3. Underlined segments are complementary to the target and bold nucleotide is specific to the G>A substitution at position 23012 resulting in the E484K amino acid change.E484K-Donor/5Phos/AACACCATTACAAGCTACGTACATGACATAATCCAAACTCATAAATTCTCGCATTTTAGATCCGTCTCCTTACAGGACACATCATCCE484-AcceptorCATCTCTAACTCTACTAAGACTTCCATTTATCAACAATAGCTGACATGTTCTAGCAGCGACAGGACACACGACAGGAGGAAAGTAACAATTAAAACCTTT
?. [LAMP Primers]
Oligonucleotides used for Loop-Mediated Isothermal Amplificationall sequences shown 5' to 3'FIPCTTTCCTCCTGTCGTGTGTCCTCCATTTATCAACAATAGCTGACBIPACCTTTAACACCATTACAAGCTACGACGGATCTAAAATGCGAGAAF3CATCTCTAACTCTACTAAGACTB3GGATGATGTGTCCTGTAAGG
?. [LAMP ]
Incubate at  for
65 °C
?. [LAMP ]
Visualize reaction tubes over a UV transilluminator. Samples containing a G>A mutation at position 23012 will exhibit fluorescence.
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Electroporation Protocol
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol may be used with electrocompetent cells prepared according to this protocol.

[BEFORE_START]
For control electroporation dilute pUC19 to 10 pg/μl with Milli-Q water.

The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.

[GUIDELINES]
Electroporation Protocol
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.

Calculation
If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)

cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)

[STEPS]
1. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
2. Place recovery SOC in 37 °C water bath.
3. Pre-warm LB-antibiotic plates at 37 °C.
4. Thaw cells on ice for 10 min or use freshly made cells.
5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
6. Flick the tube containing cells a few times to mix and add 25 µL to the microcentrifuge tubes.
7. Add 1 µL to the cells in the microcentrifuge tube.
8. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
9. Immediately add 975 µL, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
10. Rotate in the 37 °C incubator for 60 min.
11. Make appropriate dilutions.
 
12. Incubate   at 37 °C.
TITLE: Optimizing the Effect of Additives on  qPCR Detection
AUTHORS: Jonathan Phillips, Gregor Blaha

[DESCRIPTION]
Optimizing qPCR reactions has two key objectives: (1) to handle a wider range of samples, and (2) to minimize variability between replicates. [Anonymous 2009; Minas K., et al. 2011; Nolan T., et al. 2006]. Anecdotal evidence suggests that certain additives can enhance the PCR reactions and other DNA- and RNA-polymerase-based reactions, resulting in the detection of even smaller quantities of nucleic acids with higher confidence [Villalava C., et al. 2001; Rapley R., et al. 1994]. Although several mechanisms of action for these additives have been evoked, their effect on a particular assay cannot be predicted and must be determined experimentally. At the end of this protocol, we provide a table with some additives that may improve PCR-based reaction.

[STEPS]
TITLE: FBS heat inactivation
AUTHORS: Andreas Sagen

[DESCRIPTION]
The objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from serum, such as fetal bovine serum, is not required for most cell cultures but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if required for optimal cell growth. Researchers should evaluate the applicability of heat inactivation regarding their own application.

[BEFORE_START]
Clean and sanitize everything

[GUIDELINES]
Do not use heat inactivated FBS unless necessary.

[STEPS]
SECTION: Inactivation
1. Preparation
SECTION: Inactivation
1.2. Aliquot FBS into 50 mL or 100 mL units in a sterile environment
SECTION: Inactivation
2. Prepare a water bath
SECTION: Inactivation
2.1. Clean and add distilled water to an appropriate hight
SECTION: Inactivation
3. FBS inactivation
SECTION: Inactivation
2.2. Set temperature to 58 °C
SECTION: Inactivation
1.3. Prepare an volumetrically equal blank container with distilled water
SECTION: Inactivation
1.4. Equalibriate FBS and distilled water for 30 min at Room temperature
SECTION: Inactivation
1.1. Slow thaw FBS at 4 °C overnight
SECTION: Inactivation
3.1. Set FBS aliquots and distilled water blank into preheated water bath at 58 °C for 30 min Use a thermometer to track temperature in distilled water blank, and start timer when temperature reach 56 °C. Agitate aliquots every 5 min by mixing
SECTION: Inactivation
3.2. Remove aliquots from heat bath, and place FBS on ice or 4 °C for 30 min

SECTION: Inactivation
3.3. Store aliquots at -20 °C
TITLE: RNA-interference in Paramecium bursaria
AUTHORS: Ben Jenkins

[DESCRIPTION]
RNA-interference based gene knock-down in Paramecium bursaria. This collection contains protocols for:

1) Paramecium cultures for RNAi - Filtering and preparing P. bursaria cultures for downstream RNAi
2) RNAi induction - Back-dilution, induction, and creating frozen E. coli feeding stocks for downstream RNAi 
3) RNAi feeding - Daily resuspension and feeding of P. bursaria cultures
4) RNAi imaging - Imaging of P. bursaria cultures

[STEPS]
TITLE: A protocol for tissue clearing and three-dimensional imaging of human sigmoid mucosal biopsies 
AUTHORS: Pu-Qing Yuan, Tao Li, Yvette Taché

[DESCRIPTION]
This protocol was developed for tissue clearing and 3D imaging of human sigmoid mucosal biopsies by adapting and modifying the original CLARITY tissue clearing technique/immunofluorescence protocols that we established for 3D imaging and phenotyping of human colonic enteric nervous system. By using this new protocol, a piece of entire human sigmoid mucosal biopsy specimen with the volume about 6x3x1-2 mm (length x wide x thickness) can be completely cleared within 3-4 days. The 3D images and videos generated from cleared biopsy samples showed clear spatial views of nerve innervation, distributions of mast cells and enteric glial cells, and configurations of mast cells with nerve fibers in the sigmoid colonic mucosa.

[STEPS]
1.