eamag/protocols-with-steps huggingface dataset:
TITLE: Optogenetically stimulating enteric neurons in the murine large intestine.
AUTHORS: Dante Heredia, Thomas Gould

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for optogenetically stimulating neuronal subtypes in murine colonic myenteric plexus. </div></div>

[STEPS]
?. Dissection and experiments are done in the dark under infrared illumination.  A ventral midline incision is made and the whole colon is carefully excised into a Sylguard lined dissection dish containing oxygenated Krebs-ringer solution.
?. The colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon.
?. An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle.
?. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening.
?. Suture silk is used to connect three force transducers (model TST125C;  Biopac Systems, Santa Barbara, CA) to the proximal, transverse and  distal segments of the colon.
?. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6  (Biopac Systems).
?. After spontaneous colonic migrating motor complexes (CMMCs) are detected, blue light is shined continuously for 20s by a hand-held laser  (450 nm Sapphire Galaxy 3; ZBolt; Happy Valley, OR) mounted on a clamp  stand, positioned 30 cm above the preparation, or by a combination of 4 pairs of LEDs placed on either side of the colon, connected to a wire driven by an SD9 stimulator at 5Hz (20ms pulsewidth) for 5, 20 or 60 seconds.
?. After several control light stimulations, drugs are perfused and light stimulations are obtained again.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Textos utilizados para evaluar síntomas visuales y oculares asociados al uso de la lectura con smartphones. Hay cuatro textos descritivos sobre una especie animal (abejas, koalas, ornitorrincos y kanguros) junto con plantilla enla que se indican las palabras suprimidas utilizando el procedimiento cloze.</p>

[BEFORE_START]
<ul>
<li>Vamos a valorar tus síntomas visuales mientras lees en una pantalla de Smartphone utilizando diferentes lentes a una distancia de 33 cm.</li>
<li>Puede que inicialmente te notes raro o incómodo con estas lentes, es normal.</li>
<li>Las preguntas que tengas me las realizas antes de empezar la prueba.</li>
</ul>

[GUIDELINES]
<table>
<tbody>
<tr>
<td style="width: 726px;">
<table width="100%">
<tbody>
<tr>
<td>
<p><strong>ANEXO IV. INSTRUCCIONES PARA LOS PARTICIPANTES DE LAS SESIONES</strong> <strong>EXPERIMENTALES</strong></p>
</td>
</tr>
</tbody>
</table>
 </td>
</tr>
</tbody>
</table>
<p><strong><u>INSTRUCCIONES A LOS PARTICIPANTES</u></strong></p>
<ul>
<li>Se te va a presentar un texto en el móvil, consta de dos partes:
<ul>
<li>La primera, tendrás que leer en voz baja lo más rápidamente posible, tratando de comprender el contenido, hasta que aparezca la palabra “tiempo”, en ese momento me avisas.</li>
<li>Sin detenerte, sigues leyendo la segunda parte del texto en la que hay unos espacios con un número al lado, tendrás que decirme la palabra que crees que es la que falta en ese espacio, por ejemplo: “1: Casa”, si tras intentarlo no se te ocurre ninguna, dices: “Paso”.</li>
</ul>
</li>
<li>Durante la lectura debes estar en todo momento concentrado, sin hablar o mirar hacia otro lado.</li>
<li>El texto del móvil está preparado para que lo puedas mover en vertical según lo necesites, pero no se puede mover en horizontal ni agrandar el tamaño de la letra.</li>
<li>El tiempo total que vas a estar leyendo serán 25 minutos, te avisaré más o menos a la mitad y cuando queden 5 minutos para terminar. Tras la lectura tendrás que rellenar un breve cuestionario de síntomas, con unas 5-6 preguntas del anterior cuestionario que realizaste al hacer la primera parte del estudio, pero esta vez, las preguntas son referidas a lo que has sentido mientras has estado leyendo. Responde lo que realmente hayas sentido tras cada lectura, pues la gafa que finalmente te llevarás no dependerá de ello, será la de tu graduación.</li>
<li>Después dispondrás de 20 minutos de descanso en los que puedes salir de la sala.</li>
<li>Posteriormente repetiremos la prueba con otras lentes y tendrás que realizar de nuevo el cuestionario.</li>
<li>También deberás indicarme con qué gafa de las que has estado leyendo has estado más cómodo, con la primera o con la segunda.</li>
</ul>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Purification of mammalian orthoreovirus by CsCl gradient

[BEFORE_START]
Reagents
 
HO Buffer
1 mL 1 M Tris, p 7.4
5 mL 5 M NaCl
67 uL B-ME
Water to 100 mL
Filter sterilize through 0.2 micron membrane

Dialysis Buffer 
120 mL 5M NaCl
60 mL 1M MgCl2
40 mL 1M Tris, pH 7.4
water to 4 L
Filter sterilize through 0.2 micron membrane
 
1.2 g/cm3 CsCl
33.3 g CsCl
Dialysis buffer to 100 mL
Filter sterilize through 0.2 micron membrane
 
1.4 g/cm3 CsCl
67 g CsCl
Dialysis buffer to 100 mL
Filter sterilize through 0.2 micron membrane
 
 
344059         Tube, Thinwall, Ultra-Clear™, 13.2 mL, 14 x 89 mm
86703           DIALYSISTUBING SP1 8K 10MM 15M
880111          S/P CLOSURES 35MM GREEN 10/PK
 21009-284    TUBE CENT AUTOCLAV 50ML PK10 3117-0500

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>It is a crossover, double-blind, sham-controlled, pseudorandomized, and counterbalanced study at the Laboratory of Applied Neuroscience (LANA) of the Federal University of Pernambuco (UFPE). We performed a double-blind, randomized, sham-controlled crossover study with 12 healthy volunteers who underwent single sessions of rTMS (1Hz, 20Hz and Sham) and tsDCS (anodal, cathodal and Sham) associated with 20 minutes of treadmill walking. Cortical excitability was assessed by motor evoked potential (MEP) and spinal cord excitability by the Hoffmann reflex (Hr), nociceptive flexion reflex (NFR) and homosynaptic depression (HD). All measures were assessed before, immediately, 30 and 60 minutes after the experimental procedures.</p>

[STEPS]
?.
TITLE: UC Davis - Digestible Energy by Proximate Analysis
AUTHORS: Trina Knotts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Changes in fecal energy loss can result in differences in net energy balance and feed efficiency, and may result from, e.g., alterations in bile acid production, secretion or re-uptake, and changes in activities of enzymes involved in macronutrient digestion or uptake (e.g., pancreatic lipase). This assay will be coupled to food intake determinations and will involve fecal collection and feces weight measures for 48h, which can be coordinated with the meal pattern studies (catalog item D4003) or indirect calorimetry (catalog item D4007). Specific components (crude protein, dry matter, ash, and fat), will be measured by proximate analysis in fecal samples to in order to assess fat or protein absorption. </div></div>

[STEPS]
?. Mice are singly housed in clean cages with fresh food and water and low bedding.
?. Weigh mouse and “food-in” (grams of rodent diet provided at start of experiment).
?. Save several pellets of rodent diet for analysis (e.g., for energy or macronutrient content)-Store at 4C in airtight container.
?. At 24h, weigh mouse and “food –out” (grams of rodent diet remaining)
?. At the end of 48h collection period, weigh mouse and “food-out”.
?. Swap out the cage bottoms with new clean bottoms, replace feeder insert/water bottle and return animals to housing system.
?. For each cage, carefully inspect and remove each piece of bedding using tweezers to collect all feces. Inspect each pellet of feces to assure that there is no spilled food or fibers of hair or bedding attached. If significant food spillage is found in cage bottom, weigh in pre-tared weigh boat to add back to food measurement.
?. Place feces in labeled pre-weighed 1.5 ml tube.
?. Weigh tubes and record weight.
?. Subtract this value from original tube weight to determine fecal “wet” weight.
?. After collection, fecal samples are stored at -80C until analysis for bomb calorimetry and proximate analysis.
?. [Data collected:]
48h food intake 48h fecal “wet” weight fecal energy density total fecal energy loss/day fecal energy loss as a percent of energy intake breakdown of fecal energy loss: protein, fat, carbohydrate Enclose fecal/diet sample log with samples and send on dry ice to:Mouse Biology Program C/O UC Davis MMPC University of California 2795 Second Street, Suite 400 Davis, CA 95618 530-757-3333 Please include MBP/MMPC project # in reference section on package documentation and send the FedEx/UPS tracking number information to: mmpc-bodyc@ucdavis.edu
TITLE: Generating Ezrin Plasmids
AUTHORS: Shiyi Wang

[DESCRIPTION]
Generating Ezrin Plasmids

[STEPS]
1. 1. pZac2.1-GfaABC1D-BioID2-HA Construction
1.1. - **Obtain Materials**
1.2. - pZac2.1-GfaABC1D-Lck-GCaMP6f plasmid from Dr. Baljit Khakh (Addgene plasmid #52924).
1.3. - pAAV-hSyn-BioID2-Linker-Synapsin1a-HA plasmid for BioID2 sequence.
1.4. - **PCR Amplification of BioID2**
1.5. - PCR amplify BioID2 sequence from pAAV-hSyn-BioID2-Linker-Synapsin1a-HA using the following primers:
1.6. - Forward Primer: 5’-ctagcctcgagaattcaccatgttcaaaaatcttatttg-3’
1.7. - Reverse Primer: 5’-ccgggtcgactctagatgcgtaatccggtacatcg-3’
1.8. - **Insertion into pZac2.1-GfaABC1D-Lck-GCaMP6f**
1.9. - Use In-Fusion cloning (TaKaRa) to insert the PCR-amplified BioID2 sequence into the EcoRI and XbaI restriction sites of pZac2.1-GfaABC1D-Lck-GCaMP6f.
2. 2. pZac2.1-GfaABC1D-Ezrin WT-BioID2-HA and pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA Construction
2.1. - **Obtain Materials**
2.2. - pHJ421 (pEGFP-Ezrin WT) and pHJ423 (pEGFP-Ezrin T567D) plasmids from Stephen Shaw (Addgene plasmid #20680 and #20681).
2.3. - **PCR Amplification of Ezrin**
2.4. - PCR amplify Ezrin sequence from pHJ421 or pHJ423 using the following primers:
2.5. - Forward Primer: 5’-ctagcctcgagaattcaccatgccgaaaccaatca-3’
2.6. - Reverse Primer: 5’-tgaacatggtgaattccgacagggcctcgaactcg-3’
2.7. - **Insertion into pZac2.1-GfaABC1D-BioID2**
2.8. - Insert the PCR-amplified Ezrin sequence into the EcoRI restriction sites of pZac2.1-GfaABC1D-BioID2 for both Ezrin WT and Ezrin T567D variants.
3. 3. pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA Construction
3.1. - **Mutagenesis**
3.2. - Use the Q5® Site-Directed Mutagenesis Kit (NEB) to generate the Ezrin T567A mutation in pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA.
3.3. - Perform mutagenesis using the following mutagenesis primers:
3.4. - Forward Primer: CAAGTACAAGGCGCTGCGGCAGA
3.5. - Reverse Primer: TCCCGGCCTTGCCTCATG
3.6. - **Confirmation**
3.7. - Verify the presence of the Ezrin T567A mutation in the plasmid through sequencing or restriction digest analysis.
4. Notes:
4.1. - Ensure proper sterile technique and use of appropriate safety precautions during handling of plasmids and reagents.
4.2. - Perform all steps under sterile conditions to avoid contamination.
4.3. - Validate all constructs through sequencing to confirm the correct insertion or mutation.
TITLE: Labeled microtubules for single-molecule imaging
AUTHORS: John  Salogiannis

[DESCRIPTION]
This protocol is for making labeled taxol-stabilized microtubles to be used for single-molecule imaging assays by adhering to biotin slides.

[BEFORE_START]
Please take notice of the buffer preparation in the Materials section

[STEPS]
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
1. In a prechilled 1.5 mL Eppendorf tube, make a 10 µL mixture of 10 mg/mL. 
The mixture should be 80% unlabeled, 10% biotin-tubulin, and 10% 405-tubulin. 
Mix by gently flicking.
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
2. Let it sit on ice for 10 min.
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
3. Add equal volume of 2 x (10 µL). Mix by gently flicking.
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
4. Incubate at 37 °C for 30 min. Make a 1 x + 1 millimolar (mM) + 20 micromolar (µM) stock and incubate at 37 °C at the same time.
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
5. Add equal volume of prewarmed 1 x + DTT + Taxol (20 µL). Mix by gently flicking.
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
6.  Incubate at 37 °C for at least 10 min (solution will be stable for hours at this point).
SECTION: Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
7. Store in the dark at Room temperature. Should be usable for several weeks, but more aggregates will appear over time.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>Use with all Ultra Streptavidin Detection Kits.</p>
<p> </p>
<p>All steps should be done in a humidity chamber such as 926301 (http://www.biolegend.com/humidity-chamber-plus-11211.html)</p>
<p> </p>

[STEPS]
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TITLE: replication_files_Berger-et-al_COVID_GOOGLE
AUTHORS: Lawrence M Berger, Giulia Ferrari, marion.leturcq , lidia.panico , Anne Solaz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Replication files for: </span><span style = "font-weight:bold;">COVID-19 Lockdowns and Demographically-Relevant Google Trends: A Cross-National Analysis.  </span></div></div>

[STEPS]
TITLE: Gelatine bioplastic Ge01
AUTHORS: FabTextiles   Clara Davis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Gelatine bioplastic Ge01</span></div></div>

[STEPS]
?. Clean your surface and turn on hot plate.
?. Pour  of cold water into your pot of gelatine  .
240 mL
48 g
?. Measure   of Glycerol using a food scale and add it to the mixture. The amounts can be adjusted as long as the ratio remains roughly the same. Less Glycerol will result in a more brittle but harder material, more Glycerol will create a more flexible and soft sheet.
12 g
?. Stir mixed ingredients until there are no clumps and it is as dispersed as it’s going to get. Heat mixture to 95° C or to when it starts to froth. Continue stirring mixture while heating. Keep measuring the temperature and remove the heat once it’s at the right temperature or when it starts to froth. Keep stirring for another few minutes. Remove excess froth with spoon and make make sure there are no clumps in the mixture.
?. Once the mixture is ready and no clumps remain, pour it directly into the previously prepared mould. Place aluminium foil underneath mould and maybe grease mould first with vegetable oil (depending on your shape and material). Spread the liquid equally throughout the surface.
?. The drying period strongly depends on the thickness of the final product but also on room temperature and humidity. It may take up to several days until sheet is fully dry. Once the sheet is fully dry it can be removed from the mould carefully using a scalpel or thin knife.
TITLE:        ELISA for quantification of IL-11 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces.  </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">The immunoregulatory cytokine IL-41 (also known as meteorin-like protein) is expressed at high levels in the synovium of patients with psoriatic arthritis (PsA).[2]</div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div><div class = "text-block"><span>2.Onuora S. Novel cytokine, IL-41, linked with PsA.</span><span style = "font-style:italic;">Nat Rev Rheumatol</span><span>. 2019;15(11):636. doi:10.1038/s41584-019-0314-7 </span></div></div>

[STEPS]
?. An anti-human IL-11 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-11 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-11 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-11 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-11 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-11 standard dilutions and the human IL-11 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<div title="Page 1">
<p><strong>Reagents to prepare for dissociation:</strong></p>
<div title="Page 1">
<ul>
<li>
<p>4 tissue dissociator tubes</p>
</li>
<li>
<p>4 falcon tube with 20ml Percoll solution 25% in PBS minus Ca<sup>+2</sup>Mg<sup>+2</sup> (cold)</p>
</li>
<li>
<p>4 falcon tubes with Miltenyi Biotech Debris Removal Solution</p>
</li>
<li>
<p>1 bottle of PBS (-)Ca<sup>+2</sup>Mg<sup>+2</sup> (warm)</p>
</li>
<li>
<p>100ml PBS (-)Ca<sup>+2</sup>Mg<sup>+2</sup> (+)EDTA 0.5mM (warm)</p>
</li>
<li>
<p>100ml PBS (+)Ca<sup>+2</sup>Mg<sup>+2</sup> (filtered and at 37°C)</p>
</li>
<li>
<p>L<strong>iberase enzyme solution</strong> - 5.2 mg/ml stock (<a href="https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&amp;region=US" target="_blank">Sigma 000000005466202001</a>)                        0.2 Wünsch units/ml (0.385ml stock + 9.615ml Hepatozyme media)</p>
</li>
<li>
<p><strong>Stop solution</strong>: 20% FBS in PBS (-)Ca<sup>++</sup>Mg<sup>++</sup> (cold)</p>
</li>
<li>
<p><strong>Red blood cell lysis buffer</strong>: 1:10 dilution of Miltenyi Biotec RBC lysis buffer in ddH2O (MilliQ water)</p>
</li>
<li>
<p><strong>Miltenyi Biotech debris removal solution</strong></p>
</li>
<li>
<p><strong>Hepatozyme media (-)growth factors (cold)</strong></p>
</li>
</ul>
<p> </p>
<p><strong>Thumbnail image: </strong></p>
<p>https://www.tutorialspoint.com/digestion_and_absorption/digestive_glands_liver.asp</p>
</div>
</div>

[STEPS]
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TITLE: BSCI:414 Lab 3 Analyze Agarose Gel Results and Create RNAse P Primers    
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">BSCI:414 Lab 3</div></div>

[STEPS]
?. [Review Lab 2 Experiment; Analyze Data]
Review Lab 2 hypothesis and experimental setup.
?. [Review Lab 2 Experiment; Analyze Data]
Watch experiment process video, pausing to discuss.https://youtu.be/SJoLg-GCeOY
?. [Review Lab 2 Experiment; Analyze Data]
Analyze PCR fragment lengths on agarose gel. You can find a copy of the gel in our ELMS course: Files>Lab Results>Lab 2 Agarose Gel>"Lab2.agarose.gel.50.samples.jpg"Which lanes have bands? Which lanes do not? Why?Discuss cases in which F and R primers point in same direction or away from each other.
?. [Review Lab 2 Experiment; Analyze Data]
Copy results to lab notebook. Is your band size in agreement with your hypothesis?
?. [Find CDC Primers in RNAse P Gene]
In the "BSCI:414 Plasmids" under the class root folder, find gene "RNAseP_gene_NM_006413". Copy this gene to a new folder you create under "BSCI:414 Lab 3" using your name e.g. "F20_HarleyKing_Lab 3". Also give the plasmid a new name like "F20_HarleyKing_RNAseP_gene_NM_006413".
?. [Find CDC Primers in RNAse P Gene]
Navigate to the "CDC Coronavirus Real-Time RT-PCR Primers and Probes" site:https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html
?. [Find CDC Primers in RNAse P Gene]
Make primers in Benchling using the primer and probe sequences from "RNAse P". Make sure to save the primers underneath your Lab 3 folder. The primers should have a memorable name like, "F20_HarleyKing_Lab3_RNAseP-FWD".
?. [Find CDC Primers in RNAse P Gene]
Answer the following questions in your lab notebook with a table:What is the Tm of the forward primer? How many bp is it?What is the Tm of the reverse primer? How many bp is it?What is the Tm of the probe quenched with BHQ1? How many bp is it?What is the Tm of the probe quenced with 3IABkFQ? How many bp is it?
?. [Find CDC Primers in RNAse P Gene]
Copy and paste the emission spectra of the FAM probe in your lab notebook. What is its excitation/emission wavelength?
TITLE: In vitro GTPase activity
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the in vitro GTPase activity testing of purified LRRK2.

[STEPS]
SECTION: In vitro GTPase activity
1. Set up the reaction mixtures in a 96-well plate with 5 µL 20× reaction buffer (1 Molarity (M) Tris-HCl, 20 millimolar (mM) MgCl2, pH 7.5 and 2 millimolar (mM) sodium azide), 200 micromolar (µM) 2-amino-6-mercapto-7-methylpurine riboside, 0.1 U purine nucleoside phosphorylase, and 9 micromolar (µM) LRRK2 protein or 0.8 micromolar (µM) Dynamin1 or buffers for the control in separate wells.
 

SECTION: In vitro GTPase activity
2. Preincubate samples in the microplate reader for 30 min at 37 °C.
SECTION: In vitro GTPase activity
3. Add 0.5 millimolar (mM) GTP (final concentration) to initiate the reaction.
SECTION: In vitro GTPase activity
4. Immediately begin reading absorbance at  , every 1 min over 45 min at 37 °C.
SECTION: In vitro GTPase activity
5. For data analysis, subtract the last values determined before GTP was added from the corresponding values for the experimental reaction.
TITLE: NCEM Drop - Inactivation of sample on grid (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
NCEM Drop - inactivation of samples on grid

[GUIDELINES]
This procedure is used for inactivation of 3Z agents, useful if the virus has low titire.

[STEPS]
1. Adsorb 10 µL sample to grid 10 min 

2. Immerse grid in drop of 2.5 % volume   in 0.1 Molarity (M) Sorenson's phosphate buffer 10 min 

3. Wash 0.1 Molarity (M) Sorenson's phosphate buffer

4. Using filter paper wick excess buffer from grid - do not dry out, leave grid surface wet.
5. Stain   1 min 

6. Using filter paper gradually wick stain dry from grid