eamag/protocols-with-steps huggingface dataset:
TITLE: Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
AUTHORS: Yan Chen, Jennifer Gin, Christopher J Petzold

[DESCRIPTION]
This high-throughput protocol details the steps to extract protein from Gram-negative bacteria, Gram-positive bacteria, or non-filamentous fungi in 96-well plate format for quantitative proteomic workflows. This protocol uses a bench-top automated liquid dispenser but the volumes and times also apply to manual and multi-channel pipetter use. This protocol is designed for lab-based, culture conditions and synthetic community experiments where complex sample matrices are minimized. Additional sample preservation and/or protein extraction methods may be required for environmental samples (e.g., feces, soil) to minimize protein degradation and maintain sample integrity.

This protocol works best as part of a high-throughput proteomic sample preparation workflow with:

Automated Protein Quantitation with the Biomek-FX liquid handler system

and

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

[BEFORE_START]
For this protocol you will need:
- a bench-top automated liquid dispenser (e.g., Formulatrix Mantis) or manual/multi-channel pipetters
- an Eppendorf 5810R centrifuge with S-4-104 rotor or similar centrifuge

Mix at least 3 mL of NaOH/SDS buffer for final concentrations of:
- 200 mM NaOH
- 1% SDS
or use Qiagen Lysis Buffer P2 (Qiagen, Cat.#19052)

[GUIDELINES]
- All centrifuge steps use an Eppendorf 5810R centrifuge.
- You can use Qiagen Lysis Buffer P2 (Qiagen, Cat. #19052) as the alkaline-SDS cell lysis buffer.


 




[STEPS]
SECTION: Cell lysis
1. Start with 10 µL of cells per well a non-skirted PCR plate (Thermo Scientific, Cat.#AB0600).

   

SECTION: Cell lysis
3. Resuspend the cell pellet in lysis buffer on a plate mixer.

 
  
   

SECTION: Neutralization and Benzonase treatment
4. Add 2.75 µL 1 Molarity (M) to each well.

SECTION: Neutralization and Benzonase treatment
5. Add 25 µL and  0.5 µL to each well.  

SECTION: Neutralization and Benzonase treatment
6. Mix thoroughly on the plate mixer.
  
   

SECTION: Salt-Acetone protein precipitation
7. Add 200 µL to each well and let sit at room temperature for 5 minutes.

   
 

SECTION: Salt-Acetone protein precipitation
8. Centrifuge at 4000 rpm, 2 min .


SECTION: Wash and resuspend protein
11. Add 60 µL of  100 millimolar (mM) to each well to resuspend protein pellet.

   

SECTION: Salt-Acetone protein precipitation
9. Remove supernatant.

   

SECTION: Wash and resuspend protein
10. Wash protein pellet twice using 150 µL.  

 

SECTION: Wash and resuspend protein
12. Store at -20 °C until ready for Automated Protein Quantitation with the Biomek-FX liquid handler system.
SECTION: Cell lysis
2. Add  25 µL of alkaline-SDS cell lysis buffer (200 mM NaOH, 1% SDS) to each well.
TITLE: Vandy - Transverse Aortic Constriction
AUTHORS: Lin Zhong, Jeffrey Rottman, Chee Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Hypertension in man can be modeled in the mouse by introducing a constriction in the major artery (aorta) thereby obstructing outflow and increasing the afterload to the heart. This protocol describes the procedure for aortic banding aka transverse aortic constriction (TAC).</div></div>

[STEPS]
?. Mice are anesthetized with pentobarbital (50 mg.kg, IP).
?. The ventral neck and left parasternal region is shaved and disinfected with Betadine followed by 70% alcohol.
?. The mouse is positioned supine on a heating pad and a small incision is made through the skin underlying the trachea.
?. The trachea is exposed, a small puncture is made in the trachea, and endotracheal intubation is performed using a PE-50 tube.
?. The endotracheal tube is connected to a small rodent ventilator (Harvard Apparatus) for mechanical ventilation of the mouse.
?. With the use of a surgical microscope, a left thoracotomy is performed and the second intercostal space is entered using scissors and blunt dissection to expose the aortic arch.
?. A 27G blunt needle is placed parallel to the transverse aorta between the innominate and left common carotid arteries. A 7-0 ligature is placed around the transverse aorta and needle and tied.
?. The needle is removed to yield a transverse aortic constriction (TAC) of ~0.4 mm in diameter. In sham mice, the entire procedure is identical except for the ligation of the aorta.
?. The chest is closed in layers with 7-0 sutures.
?. The mouse is gradually weaned from the ventilator to resolve any possible pneumothorax.
?. Once spontaneous respiration resumes, the endothracheal tube is removed and the trachea is closed with 8-0 suture. The skin is then closed with 6-0 suture.
?. The mouse is maintained on the heating pad until fully recovered from anesthesia.
?. Buprenorphine is administered SC immediately following surgery and every 8-12 hr for 72 hr.
?. One day following the TAC procedure, the mouse is subjected to Doppler echocardiography (Vevo2100) to determine the degree of stenosis induced by the ligation.
TITLE: EA.hy 926 RhoA Fret with PAsssr in Flow Cytometry
AUTHORS: Cody Roberts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RhoA Activation test with PAsssr and C3</div></div>

[STEPS]
?. Split EA.hy 926 RhoA Sensor Cells into each well of a 6 well Plate. (Seeding density is about 300,000 cells and 1.2 million at confluence. Plate around 500,000 to have cells ready to run the next day. (You will want about 75-95% confluence the day of the experiment.)
?. Allow Cells to grow to 75%-95% confluence.
?. Day of experiment, treat cells for 4 to 8 hours depending on the conditions you want. PAsssr for CMG2 targeting should be treated at a concentration of  200pM , but use PAsssr at the begining to test for the effect because it might be a subtle change. C3 (RhoA inhibitor is at 1ug/ml I believe. I would double check that number.) You will want a no treatment well, RhoA inhibition well (C3), and a CMG2 inhibition well (PAsssr). Unlableled PAsssr can be found in the frosted -20 in a box labeled PA.
?. Morning of, change the media to DMEM (Serfum Free) supplimented with VEGF. I am not sure what the working concentration Jeremy used for Migration, but I would check his notes and use the same concentration. VEGF can be found in the -80. I believe you will want to use   BSA aswell, but I would again check jeremy's notes on that.
?. After the treatment times is over, asparate the media and add 0.5ml of Trypsin to each well.
?. Incubate 10 minutes
?. Move tyrpsin plus cells to 1.5 ml tubes and spin at 200g for 10 minutes.
Centrifuge: 200 34, 10 min, 24 10
?. Asparate media and add 200ul of prechilled PBS. Pipet up and down to break up any clumped cells.
?. Add the 200 ul of PBS plus cells to a 96 well plate and leave on ice. Samples are ready to be run in flow.
TITLE: microC: A 3D virtual microenvironment for perturbation biology
AUTHORS: Dimitrios Voukantsis, Kenneth Kahn, Martin Hadley, Rowan Wilson, Francesca M Buffa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">microC is a multiscale virtual microenvironment for perturbation biology. It enables experiments that link genotype to phenotype taking into account the surrounding microenvironment. microC has a modular architecture that enables a wide variety of experiment. Furthermore, it offers easy access to advanced computational modelling and supercomputing resources to the wider scientific community.</div><div class = "text-block">microC combines agent-based and gene network modelling and uses partial differential equations to simulate interactions among cells. </div><div class = "text-block">The tool itself does not require any installation on the user’s machine, as it can be accessed using a web browser. Experimental results and data are also available via a web interface. This protocol describes the process of preparing and submiting an experiment with microC, and interpreting the simulation results</div></div>

[STEPS]
?. [Accessing microC]
microC is available via microc.org. During the first visit this step might take several seconds. This will not be the case in future visits.
?. [Preparing an experiment]
Experiments are specified via the microC main web page (Figure 1). The user may set a number of numerical parameters via sliders, specify the gene network, mutations, and parameters that control diffusion (please, refer to the microC-detailed-protocol.pdf for more details). microC modules are enabled through the specification files. For example setting parameters for Oxygen or any output node within diffusion-parameters.txt, will automatically enable a diffusion module for this substance. The "Run expreriment" buttom submits the expreriment to the computing cluster.
?. [Submitting an experiment]
Upon submission of the experiment (“Run Experiment” button, Figure 1) the user receives a link (Figure 2) that provides information about the status of the submission and the log file, where potential errrors are shown. Once the experiment has finished the same link will contain the results (that gradually become available in batches 16 replicates). Optionally, the user will be notified by email.
?. [Evaluating the results]
The results page displays data across all replicates and permits the viewer to inspect any of the replicates in detail. An animation of all replicates is displayed in the initial page along with averaged and detailed data for each of the experimental replicate (Figure 3). A more detailed presentation of the results page is available in the documation page of microc (http://microc.org), or microC-detailed-protocol.pdf .
TITLE: Mix and go competent cells
AUTHORS: Ben Kuipers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mix and Go protocol for the preparation of competent E. coli cells. (This protocol is according to Zymo Research mix and Go protocol).</div></div>

[STEPS]
?. Use 0.5 ml of fresh, overnight E. coli culture grown in LB to inoculate 50 ml ZymoBroth™ or SOB medium in a 500 ml culture flask. Shake culture vigorously (150 - 250 rpm) at the appropriate temperature* until the OD600nm is 0.4 - 0.6. (Buffer Preparation Prior to Harvesting the Cells: The Wash and Competent Buffers are provided as 2X stock solutions. They need to be diluted to 1X by adding an equal amount of Dilution Buffer. To prepare 5 ml of 1X Wash Buffer: Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock Wash Buffer. To prepare 5 ml of 1X Competent Buffer: Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock Competent Buffer. Please keep these freshly prepared 1X Buffers ice cold. These 1X Buffers are good for 2 days at 0-25°C. It is important that each step of the following procedure should be done on ice or at 0-4°C.)
?. Transfer the culture from Step 1 to ice. After 10 minutes, pellet the cells by centrifugation at 3,000 - 3,700 rpm (i.e., 1,600 - 2,500 x g) for 10 minutes at 0 - 4°C.
?. Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash Buffer. Re-pellet the cells as in Step 2.
?. Completely remove the supernatant and gently resuspend the cells in 5 ml ice-cold 1X Competent Buffer.
?. Aliquot (on ice) 0.1-0.2 ml of the cell suspension into sterile microcentrifuge tubes. Cells are now ready for transformation with DNA or can be stored below -70°C for transformation at a later time.
TITLE: Startle induced negative geotaxis (SING)
AUTHORS: Natalie Kaempf, Uli Pech, Patrik Verstreken

[DESCRIPTION]
This protocol describes how to perform the Startle induced negative geotaxis (SING) assay. SING defects in parkinsonism fly models have previously been shown to be caused by dopaminergic impairments.

[STEPS]
SECTION: Startle induced negative geotaxis (SING)
2. set a specific time period during the day (e.g. Zeitgeber 3-5) to assess negative geotaxis behavior, which will be kept the same for all following experiments
SECTION: Startle induced negative geotaxis (SING)
3. flies are transferred into the first tube of the apparatus without prior anesthesia and allowed to adjust
for some minutes to experimental environment (24±1ºC, 40-60% humidity in behavior chamber, note down the environmental condition and time for every genotype)
SECTION: Startle induced negative geotaxis (SING)
4. tap the flies down five times and allowed to climb up for 30 s

SECTION: Startle induced negative geotaxis (SING)
5. after these 30 s the flies that reached the upper tube are moved to the next tube
SECTION: Startle induced negative geotaxis (SING)
6. repeat the procedure 4 times
SECTION: Startle induced negative geotaxis (SING)
7. count the number N of (male/female) flies with the corresponding genotype per tube to calculate the SING score, which is normalized to the mean SING score of the control. 

SECTION: Startle induced negative geotaxis (SING)
8. SING score = ((N1 *0) + (N2 *1)+ (N3 *2)+ (N4 *3)+ (N5 *4))/ 4(N1 + N2 + N3 + N4 + N5 )
Nk = number of flies in the kth tube
 
 
SECTION: Startle induced negative geotaxis (SING)
1. for the describtion of the SING apparatus please refer to: 
Benzer, Seymour. 1967. “BEHAVIORAL MUTANTS OF Drosophila ISOLATED BY COUNTERCURRENT DISTRIBUTION.” Proceedings of the National Academy of Sciences 58 (3): 1112–19. https://doi.org/10.1073/pnas.58.3.1112.

Inagaki, Hidehiko K, Azusa Kamikouchi, and Kei Ito. 2010. “Methods for Quantifying Simple Gravity Sensing in Drosophila Melanogaster.” Nature Protocols 5 (1): 20–25. https://doi.org/10.1038/nprot.2009.196.
TITLE: Long term effect of Aldicarb on C. elegans
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Aldicarb is a cholinesterase inhibitor which prevents the breakdown of acetylcholine in the synapse​</li><li style = "counter-reset:ol0;list-style-type:disc;">Reported to paralyze C. elegans​</li><li style = "counter-reset:ol0;list-style-type:disc;">Strains of interest believed to be resistant to the paralytic effect​</li><li style = "counter-reset:ol0;list-style-type:disc;">Strains: OW1601 & OW1603</li></ul></div></div>

[STEPS]
?. Bleach synchronize the worms on a Friday
?. Following Monday, seed 4 maintenance plates with OP50 and dry under hood
?. Add 150ul of Aldicarb solution to the 4-maintenance plate to reach a final concentration of 3uM and leave to absorb overnight
?. The following day (Tuesday), refeed the arrested L1s on the treatment plates and normal OP50 seeded plates i.e. OW1601 L1s refed on 2x3uM aldicarb plate + 2xnormal OP50 seeded plate. Same for OW1603. So, each strain will have two control plates and two treatment plates- Total 8 plates
?. Keep one set of plates at 25C and the other set at 20C
?. The day before tracking (Thursday) add 35ul of 300uM AK to 24 imaging plates
?. Also, seed those drugs treated 24 imaging plates and additional 24 non-treated imaging plates with 50ul of 1:10 OP50 and leave to dry O/N
?. On the day of tracking (Friday), transfer 5 worms onto each imaging plate and image for 15mins on PhenixTrack 8 sets: (2 treatment sets + 2 control sets with no treatment) x 2 growth temperatures
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll <em>a</em> fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; <em>Thalassiosira pseudonana </em>CCMP1335, chain-forming <em>Skeletonema marinoi </em>RO5A and <em>Coscinodiscus radiatus</em> CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d<sup>-1</sup>) using an RStudio script, SinkWORX.</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to knockout mammalian non-coding RNA genes through excision of an epigenetic transcriptional start site (TSS) signature. To this end two guideRNAs are cloned onto the pX458 CRISPR vector to induce DNA cleavage right up- and downstream of the TSS element.</p>
<p>The protocol describes how to generate homozygous knockout cell clones. Optionally, successful target DNA excision by the dual guideRNA pX458 construct may be pre-validated by transfection into an easy-to-transfect cell line (e.g. Hek293) and genomic PCR without prior single cell clonal expansion.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background </strong></p>
<p>Vesicovaginal fistula is a debilitating condition resulting from prolonged obstructed labor. Globally, at least 2 million women are estimated to be living with fistula with about 50,000 to 100,000 new cases each year. More than 90% of the global burden of fistula is in sub-Saharan Africa and South East Asia. Traditional management of fistula requires surgical repair. However, many women either do not have access to surgery, or access to surgery is delayed. These categories of patients have few or no options to control the constant urinary leakage.  The vaginal menstrual cup is an insertable medical device approved for use in Europe and North America to collect menstrual flow. Because it collects fluid drainage from the vagina, it could also be used to control urine leakage in women with vesicovaginal fistula.  While surgical management of fistula would remain the gold standard in treatment, the menstrual cup could be an alternative for women who do not have access to surgery or are poor surgical candidates.</p>
<p><strong> </strong></p>
<p><strong>General Aim </strong></p>
<p>To evaluate the menstrual cup for short-term non-surgical management of vesicovaginal fistula.</p>
<p> </p>
<p><strong>Methodology </strong></p>
<p>This study will examine the reduction in urinary leakage for women with vesicovaginal fistula who use the cup over a 2 hour period. Fistula patients recruited after community durbars  will be invited to the hospital and offered enrollment. They will wear a sanitary pad for 2 hours and the pad will be weighed. The patients will then receive counseling on how to insert the cup in the vagina and will wear both the device and a sanitary pad for 2 hours. The second pad will also be weighed. The amount of urine leaked with and without the cup will be compared using a paired t-test. Addtionally, the women will answer a short questionnaire on the acceptability of the cup, and a physical exam will be carried out to assess safety with cup use. A purposeful sample of the patients will be recruited for in-depth interviews to share their experiences of coping with VVF and their acceptability of the menstrual cup device.</p>
<p><strong><br /> </strong></p>
<p><strong> </strong></p>
<p><strong>Expected Outcome </strong></p>
<p>The knowledge gained from this study may offer alternative temporizing management options for fistulas, especially for women who fail or are awaiting surgery, women who cannot access surgery, or are poor surgical candidates.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is based on RT Noble and JA Fuhrman (1998) Aquatic Microbial Ecology 12:113-118: Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.

[GUIDELINES]
<p><strong>Old Procedure<br /><br /></strong>1.) &nbsp;Make dilution of virus prep in 0.02&micro;m filtered seawater.<br /><br />2.) &nbsp;Prepare staining droplet using 2.5-4&micro;l SYBR green or gold (10,000x stock diluted to 10% with 0.02&micro;m filtered Q-water) mixed with 97&micro;l 0.02&micro;m filtered Q-water and place cover over droplets to keep dark.<br /><br />3.) &nbsp;Prepare 25 mm SYBR towers:&nbsp; 0.02&micro;m Anodiscs, careful not to break membrane (can use 0.2&micro;m backing filter underneath if desired).&nbsp; Filter sample onto membrane using vacuum.&nbsp; Rinse tower in 1L Q-water in between samples and blot onto paper towel to dry.<br /><br />4.) &nbsp;Remove membrane carefully and set down on the edge of the droplet of diluted SYBR stain, letting the SYBR float under the filter.&nbsp; Incubate RT, dark for 15 min.<br /><br />5.) &nbsp;Pick up filter off drop of stain and blot by passing over kimwipe two times.&nbsp; Place membrane, cell side up, onto microscope slide.&nbsp; Pipet 32&micro;l 0.1% phenylenediamine (made in PBS/50% glycerol) on 22mm square coverslip.&nbsp; Invert over filter on microscope slide and press down to spread mounting medium.<br />6.) &nbsp;Place slide at &ndash;20<sup>o</sup>C to enhance fluorescence.&nbsp; Read slides using 100x oil immersion objective and inverted fluorescent microscope.</p>

[STEPS]
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TITLE: p53 immunohistochemistry protocol
AUTHORS:  Ratha-Korn Vilaichone, Natsuda Aumpan

[STEPS]
?. Deparaffinize and rehydrate sections:Incubate 3 times in xylene for 5 minutes each.Incubate 3 times in 100% Ethanol for 5 minutes each.Hydrate by placing in 90%, 80% ethanol for 5 minutes each.Immerse slides in tap water for 5 minutes.
?. Sodium Citrate Antigen Retrieval:a. Place rack in Sodium Citrate buffer (10 mM, pH 6.0) in a glass beaker.b. Autoclave for 10 minutes at 120 °C.c. Immerse glass beaker in tap water to cool.d. Immerse glass slides in tap water for 5 minutes.
?. Block endogenous peroxidases:a. Soak slides in 3% H2O2 for 5 minutes at room temperature.b. Wash once with PBS.
?. BlockingShake and wipe off excess PBS. Circle all sections with a DAKO pen. Add blocking buffer (10% normal goat serum) to each section immediately, so that the sections don’t dry out. Don’t touch sections with tip. Incubate for 20 minutes at room temperature in a humidified chamber. Wash in PBS for 5 minutes.
?. Primary antibodyDilute primary antibody (anti-p53) in antibody dilution buffer (1:800).Add 50-75 μl per section and incubate overnight at 4 °C in a humidified chamber.
?. Drain primary antibody off section. Wash slides 3 times in PBS for 5 minutes.
?. Dilute biotinylated secondary antibody. Add 50-75 μl per section and incubate more than 30 minutes at room temperature in a humid chamber.
?. Drain secondary antibody and wash slides 3 times for 5minutes each in PBS.
?. Peroxidase visualization:Mix 1 drop Vectastain "A" and 1 drop Vectastain "B" in 5 ml PBS (for 20 slides) as per kit instructions at the beginning of the secondary antibody incubation (30-60 minutes before use).Incubate 10-20 minutes in a humidified chamber. Rinse with buffer as before.
?. Incubate in fresh DAB solution.(30 mg DAB + 30 μl 30% H2O2 in 150 ml 0.05 M Tris pH 7.6)
?. Stop the reaction by washing in PBS.
?. Counterstain with hematoxylin.
?. Dehydrate sections:Incubate once in 70% Ethanol for 5 minutes.Incubate 3 times in 100% Ethanol for 5 minutes each.Incubate 3 times in xylene for 5 minutes each.
?. Coverslip slides using Permount (xylene based).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<p>Basic protocol to plate Human Embryonic Kidney 293 (HEK293) cells in to 12 well plates. This protocol can be modified easily to plate in different volumes and concentrations. </p>
</div>
</div>

[BEFORE_START]
<p>Make complete DMEM:</p>
<p> </p>
<table width="312">
<tbody>
<tr>
<td><strong><em>Reagent</em></strong></td>
<td><strong><em>Volume</em></strong></td>
</tr>
<tr>
<td>DMEM</td>
<td>432.5 mL</td>
</tr>
<tr>
<td>FBS</td>
<td>50 mL</td>
</tr>
<tr>
<td>Pen/Strep</td>
<td>5 mL</td>
</tr>
<tr>
<td>HEPES (1M, pH 7.4)</td>
<td>12.5 mL</td>
</tr>
</tbody>
</table>
<p> </p>

[STEPS]
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