eamag/protocols-with-steps huggingface dataset:
TITLE: Growth Curves of S. elongatus under salt stress and low carbon
AUTHORS: Akashdutta  

[DESCRIPTION]

When liquid media is inoculated with bacteria and the cell population is counted at intervals, it is possible to plot a typical bacterial growth curve that shows the increase in the number of cells over time. Such growth curves show four distinct phases of growth:

Lag phase: There is slow growth or lack of growth due to the physiological adaptation of cells to culture conditions or the dilution of exoenzymes (due to initial low cell densities).
Log or exponential phase: Optimal growth rates are seen in this phase. Cell numbers double at discrete time intervals known as the mean generation time.
Stationary phase: During this phase, the growth (cell division) and death of cells occur at the same rate, resulting in the number of cells being constant. The reduced growth rate is usually due to a lack of nutrients and/or a buildup of toxic waste constituents.
Decline or death phase: Here, the death rate exceeds the growth rate, resulting in a net loss of viable cells.

This is one of the simplest methods used to analyze trends in growth because it uses a spectrophotometer to track changes in the optical density (OD) over time. In other words, as the number of cells in a sample increases, the transmission of light through the sample will decrease.

Growth curves for certain freshwater cyanobacterial species are carried out under salt stress to account for sucrose production in the particular strain. This is because sucrose is naturally produced intracellularly in these strains to balance the osmotic pressure of a saline environment.


[STEPS]
1. Take 100 mL culture in BG-11 . Add 150 milimolar (mM) Nacl and note the time of addition.

2. 3 hours after salt acclimation, take 2 1 mL  samples from each flask in 2 microcentrifuge tubes

3. Use one set of samples to take the OD measurements
4. Take the other set of samples and centrifuge at 4000 rpm, 7 min, 25 °C 

5. Take the supernatant in fresh microcentrifuge tubes and store it at -20 °C . Discard the pellets.

6. Take the readings every three hours until the OD730  value reaches 1.0
TITLE: mynetsohbet
AUTHORS: sohbet 

[STEPS]
?. http://www.sohbet.world  Eski mynet sohbet odaları omegla sohbet yazılı omegla chat 18 sohbet odaları yaş grupları akıllı telefon ve tablet uyumlu üyelij gerekmeyen ücretsiz resimli sesli görüntülü sohbet ve cgat seçenekleri.
?. 
?.
TITLE: Differentiation of SH-SY5Y cells
AUTHORS: Shenjie Wu, Nancy C. Hernandez Villegas, schekman

[DESCRIPTION]
This protocol describes a standard procedure to differentiate SH-SY5Y cells into dopaminergic neurons using retinoic acid  

[STEPS]
SECTION: Differentiation of SH-SY5Y cells
1. SH-SY5Y neuroblastoma cells were maintained in DMEM supplemented with 1× nonessential amino
acid (NEAA), 1× sodium pyruvate, and 10% FBS. 
SECTION: Differentiation of SH-SY5Y cells
2. Differentiation was induced by lowering the FBS in culture medium to 1% plus 10 μM RA. 
SECTION: Differentiation of SH-SY5Y cells
3. Cell medium was replaced each 3 days to replenish RA.
SECTION: Differentiation of SH-SY5Y cells
4. Cell morphology was monitored by microscopy and experiments on SH-SY5Y cells were performed from D6 of differentiation.
TITLE: DALEX NonTox PAGE Stain
AUTHORS: David Frommholz, Alexandra Ehl, Nadine Stefanczyk

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">DALEX NonTox PAGE Stain is a non-toxic Commassie based protein dye for the detection of bands in SDS and Native PAGE. The easy and fast staining procedure does not require time consuming destaining of the gels and is compatible with mass spectrometry.  </div><div class = "text-block">First protein bands are visible within minutes and in most cases gel documentation can be done after 15-30 minutes of staining.</div><div class = "text-block">The sensitivity depends on the gel’s lane width and on how “packed” the protein band is, which depends on various factors such as acrylamide percentage of separation/stacking gel and MW of the protein. Under ideal conditions the detection limit can be as low as 5 ng per band.</div></div>

[STEPS]
?. [Washing]
Remove the gel from the cassette and place it into a tray e.g. a weighing pan.Immerse the gel in distilled water (50 ml for an 8 x 8 cm gel) and microwave for 30 s at 500 W.Incubate with gentle shaking for 5 minutes and discard the water afterwards.Repeat two more times.
Native PAGE gels do not require washing because they do not contain SDS. In this case you can skip the washing step.
?. [Staining]
Add enough DALEX NonTox PAGE Stain to completely cover the gel ( 25 ml for an 8 x 8 cm gel).Microwave for 10 s at 500 W.
?. [Staining]
Incubate with gentle swirling or rocking for at least 15 minutes or until the desired stainig intensity is reached.
?. [Washing]
Remove your gel from the gel plates/cassette in place in a clean bowl or container, e.g. a weighing pan. Add dest. water to completely cover the gel. Microwave for 30 s at 500 W. But do not boil!Incubate for 5 minutes with gentle shaking, discard the water.Repeat twice.
[Microwave 500 V]
[Gentle shaking]
50 ml water for washing a 8 x 8 cm gel works well.
?. [Staining]
Cover the gel with DALEX NonTox PAGE Stain, microwave for 10 s at 500 W and incubate on a shaker until the desired intensity is reached.
~25 ml staining solution for an 8 x 8 cm gel works well.
?. [Staining]
Pour off the staing solution and rinse the gel with water twice. To increase the contrast of the gel, the background can be reduced by washing the gel in water.
?. [Staining]
Pour off the stainging solution and rinse the gel with water twice. To increase the contrast of the gel, the background can be reduced by washing the gel in water and heating in a microwave as described in step 1.
TITLE: Overall protocol for top-down CZE-MS/MS of human spleen tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down CZE-MS/MS data on human spleen tissue

[STEPS]
SECTION: Tissue Collection
1. Spleen tissue was provided by University of Florida TMC. Tissue was collected and prepared according to the following: 


 


SECTION: Sample Preparation
2. 
 

SECTION: Data Acquisition
3. 
 

SECTION: Data Analysis
4.
TITLE: T4H protocol
AUTHORS:  Ophir Auslaender, kalle.levon 

[STEPS]
?. Clean Au chip: insert in piranha  solution (75% H2SO4, 25% H2O2), wash with DI water, dry with nitrogen gas.
?. Mix the solution for imprinting in 19/1 volume ratio: 19 for the analyte, 1 for the monolayer former at self-assembled monolayer (SAM) concentration.
?. Place Au chip in the imprinting solution. Incubate overnigth RT.Wash multiple times with 3M NaCl solution.Leave overnight in DI water.
?. At this point the chip can be sealed for future use. Prior to measurement chip is inserted in PBS for 2 hours.
?. The chip is connected as the working electrode in a potentiostat circuit.A reference electrode is added to the reaction vessel and recording of the open-circuit potential (OCP) is started. When the voltage stablilizes, the analyte is added.
?. Adding the analyte shifts the OCP if the analyte is recognized by the imprinted working electrode.In the calibration phase, controlled serial dilutions of analyte are added, and resulting OCP values are recorded.  The relationship between analyte concenteration and OCP is established from these calibration measurements, and is used for the detection of unknown analytes.
TITLE: Free radical scavenging activity  
AUTHORS: Jorge Carlos Ruiz Ruiz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Free radical scavenging assay was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical test. To each aqueous extract 3 mL of 0.1 mM solution of DPPH in methanol was added (Fermont, Monterrey). Tubes were shaken vigorously and allowed to stand for 30 min at room temperature in the dark. Absorbance was measured at 517 nm. Distilled water was the control and ascorbic acid served as the standard. Free radical scavenging activity was expressed as inhibition percentage and was calculated using the formula (A0 − A1)/A0 × 100, where A0 was the control absorbance and A1 was the sample absorbance. All tests were performed in triplicate and antioxidant activity was quantified by a regression analysis of percentage of free radical scavenging (%) versus phenolic compound concentration in the aqueous extract; this was defined as an IC</span><span style = "vertical-align:sub;">50</span><span> value, which is the amount of antioxidant necessary to decrease the initial DPPH radical concentration by 50%. Ascorbic acid was used as control.</span></div></div>

[STEPS]
?. To each aqueous extract 3 mL of 0.1 mM solution of DPPH in methanol was added. Tubes were shaken vigorously and allowed to stand for 30 min at room temperature in the dark.
?. Absorbance was measured at 517 nm. Distilled water was the control and ascorbic acid served as the standard.
?. Free radical scavenging activity was expressed as inhibition percentage and was calculated using the formula (A0 − A1)/A0 × 100, where A0 was the control absorbance and A1 was the sample absorbance.
?. Antioxidant activity was quantified by a regression analysis of percentage of free radical scavenging (%) versus phenolic compound concentration in the aqueous extract; this was defined as an IC50 value, which is the amount of antioxidant necessary to decrease the initial DPPH radical concentration by 50%. Ascorbic acid was used as control.
TITLE: Top2 Chromatin Accessibility by Etoposide Cross-linking
AUTHORS: Jacob Kirkland

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">The best way to determine TOP2 specific accessibility to chromatin is via etoposide treatment, which leads to a covalent cross-link between TOP2 proteins and DNA, that can only occur where TOP2 proteins have access to DNA. TOP2 cross-linking to DNA has previously been shown to require DNA accessibility and chromatin regulators that alter DNA accessibility (Dykhuizen and Hargreaves et al. Nature 2013, Miller et al. NSMB 2017).</span></div></div>

[STEPS]
?. Cell Culture:Trypsinize and count cellsTransfer 0.75e6-1e6 cells to (4) different eppendorfsSave (1) eppendorf for your input sample for step 4Bring volumes to 1ml with media (If volume is already more than 1ml you can transfer to a 15ml conical and bring volume up to known amount where all cell lines are at the same volume)
?. Etoposide Cross-linkingAdd Etoposide (1:1000; final concentration of 100uM) for 0, 15 and 60 minutes    Add to 60' sample first, and then 15' sample 45' laterRotate at RT for 60'
?. WashesSpin at 500g for 4 minutes at 4°CWash with PBSSpin at 500g for 4 minutes at 4°CRemove suppernatant
?. Buffer A -- Making NucleiFor all samples + InputResuspend in 1 ml  Buffer A + Protease Inhibitors (1:1000) + DTT (1:1000; final concentration:)Incubate on ice for 10'Spin at 300g for 4 minutesAspirate out suppernatantSpin breifly and remove remaining suppernatant
?. High salt RIPA extractionResuspend nuclei in 50-100ul High salt RIPA with 500mM NaClIncubate on ice for 20'Spin 12,000g for 5' at 4°CRemove suppernatant (can save if you want)Spin breifly and remove remaining suppernatant
?. Solubalize chromatin pelletResuspend in 50ul 1% SDS RIPA + PIs + DTT + Benzonase (1:200)    Be sure to fully resuspend pellet by pipetting up and down ~20 timesIncubate at room temp for 10'Incubate on ice for 20'Spin 12,000g for 3 minutes and transfer suppernatant to a new tube (there should not be much if any of a visible chromatin pellet after spinning. If there is then try resuspending in a larger volume)
?. Proceed to Western Blot adding equal volumes of each sample
TITLE: Conditioned Media Concentration with Amicon Ultra Centrifugal Filters
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Conditioned media has to be concentrated for downstream processing. To do this, complete media was removed the day before the study endpoint from cultured cells/tissue, and were washed with serum-free media. Cells/tissue were then incubated with serum-free media for 24 hours. After 24 hours, the conditioned media was collected from cultured cells/tissue and concentrated using centrifugal filters.

[STEPS]
1. Insert the Amicon Ultra 0.5 mL Centrifugal Filter into one of the provided microcentrifuge tubes.
2. Add up to 0.5 mL of conditioned media to the filter device and cap it.
3. Place the capped filter device into the centrifuge rotor and align the cap strap toward the center of the rotor; counterbalance with a similar device.
4. Spin the device at 12,000 × g for approximately 10 minutes.
5. Repeat steps 2 to 4 until the entire volume of conditioned media is concentrated down to ~50 μL. 
6. Remove the assembled device from the centrifuge and separate the filter device from the microcentrifuge tube.
7. To recover the concentrated solute, place the Amicon Ultra filter device upside down in a clean microcentrifuge tube. Place in the centrifuge and align the open cap towards the center of the rotor; counterbalance with a similar device. 
8. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device to the tube.
9. Continue with downstream processing or store at -80°C.
TITLE: Supplement Figure
AUTHORS: Lijun Zhang, lijun zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = ":;"> </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;:;"> </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">supplement figure 1</span></a><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;"> </span></a></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bdkba.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bdj8a.png" /></div></div>

[STEPS]
TITLE: Isolation of  mononuclear cells using Septmate    
AUTHORS: Ajit N Shah

[STEPS]
?. Add density gradient medium to the SepMateTM tube by carefully pipetting it through the central hole of the SepMateTM insert. Refer to step 2 for required volumes. The top of the density gradient medium will be above the insert.NOTE: Small bubbles may be present in the density gradient medium after pipetting. These bubbles will not affect performance.
?. Dilute sample with an equal volume of PBS + 2% FBS. Mix gently.For example, dilute 5 mL of sample with 5 mL of PBS + 2% FBS.
?. Keeping the SepMateTM tube vertical, add the diluted sample by pipetting it down the side of the tube. The sample will mix with the density gradient medium above the insert.NOTE: The sample can be poured down the side of the tube. Take care not to pour the diluted sample directly through the central hole.
?. Centrifuge at 1200 x g (see Notes) for 10 minutes at room temperature, with the brake on.NOTE: For samples older than 24 hours, a centrifugation time of 20 minutes is recommended.
?. Pour off the top layer, which contains the enriched MNCs, into a new tube. Do not hold the SepMateTM tube in the inverted position for longer than 2 seconds.NOTE: Some red blood cells (RBCs) may be present on the surface of the SepMateTM insert after centrifugation. These RBCs will not affect performance.NOTE: To reduce platelet contamination in the enriched MNCs, pipette off some of the supernatant above the MNC layer before pouring.
?. Wash enriched MNCs with PBS + 2% FBS. Centrifuging at 300 xgfor 8 minutes at RT.
?. [ ]
ABC1SEPMATETM TUBEINITIAL SAMPLE (mL)DENSITY GRADIENT MEDIUM (mL)2150.5 - 4.04.5315&gt;4-53.54504 - 1715
ABC1SEPMATETM TUBEINITIAL SAMPLE (mL)DENSITY GRADIENT MEDIUM (mL)2150.5 - 4.04.5315&gt;4-53.54504 - 1715
?. [ ]
?. Add  ACK Lysis buffer and incubate for 3 minutes. Add 3-4 volumes of PBS + 2% FBS. Centrifuging at 400 xg for 5 minutes at RT.
?. Re-suspend in desired volume of media/buffer and perform cell count.
?. PMBC are ready for downstream process.
?. Wash enriched MNCs with PBS + 2% FBS. Centrifuging at 400 xg for 5 minutes at RT.
TITLE: A randomized clinical trial of exenatide onT2D
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Among the Chinese adults with diabetes, the proportion of overweight is 41.0%, the proportion of obesity is 24.3%, and the proportion of abdominal obesity is as high as 45.4% in 2008, which shows an increasing trend in recent years. Clinical studies have demonstrated that Asian people have higher levels of visceral fat than European and American populations. Obesity, especially abdominal obesity is closely associated with Diabetic Nephropathy (DN). exenatide improves glycemic control and reduces body weight in patients with T2DM. In animal studies, </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">exenatide </span></a><span>can ameliorate albuminuria and improve kidney function. However, only a few studies evaluate the microalbuminuria in human. This study aims to investigate the effect of </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">exe</span></a><span>natide on urinary albumin, obese indices and glucose control in overweight/obese patients with type 2 diabetes.</span></div></div>

[STEPS]
TITLE: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
AUTHORS: cgarcia

[DESCRIPTION]
La solicitud de admisión será on-line y permanecerá abierta durante todo el curso académico.
Preinscripción  on-line
Podrán preinscribirse quienes cumplan los requisitos de acceso a los programas de doctorado y también los estudiantes que no hayan acabado los estudios que dan acceso al Doctorado. En este caso su admisión está condicionada a obtener la titulación antes de formalizar la matrícula.
NOTA: Es conveniente consultar en la web del programa de doctorado elegido, por si existiera algún criterio específico de admisión antes de proceder a la preinscripción.

[STEPS]
SECTION: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
1. Realizar la preinscripción on-line, anexando a la misma (exclusivamente en formato pdf) los documentos exigidos en la aplicación, que son:

·Documento acreditativo de su identidad y nacionalidad.
·Títulos que dan acceso al doctorado.
·Certificado académico de los títulos de acceso, con nota media en base 10.
·Aquellos otros documentos específicos reseñados en el programa de doctorado en el que solicita admisión.
·Los estudiantes extranjeros ajenos al EEES, además, presentarán una certificación indicando que los títulos aportados les facultan, en el país expedidor del título, para el acceso a estudios de doctorado.
·Currículum vitae con justificantes acreditativos de los méritos.
·Carta de motivación donde se indique: línea de investigación de interés y profesor de contacto en el programa, si existe.
SECTION: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
2. Indicar la dedicación al programa (tiempo completo o tiempo parcial). Aportando la documentación necesaria en los casos en que se pretenda realizar a tiempo parcial.
SECTION: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
3. Aportar la documentación original, o copias legalizadas, en la Secretaría de la Escuela Internacional de Doctorado de la Universidad Católica San Antonio de Murcia, Avda. de Los Jerónimos s/n 30107, Guadalupe, Murcia (personalmente o vía correo postal).
SECTION: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
4. Si toda la documentación es correcta, su solicitud será admitida a trámite y pasará a ser evaluada por la Comisión Académica del Programa correspondiente.
SECTION: PROCEDIIENTO PARA LA SOLICITUD DE ADMISIÓN A UN PROGRAMA DE DOCTORADO DE LA EIUCAM (PREINSCRIPCIÓN)
5. Esta circunstancia la podrá comprobar a través de la plataforma de solicitud de preinscripción on-line.
TITLE: Nuclease Test (OpenVent polymerase, PCR Master Mix, DNA loading dye)
AUTHORS: Nadine   Mowoh, Stephane  Fadanka

[DESCRIPTION]
Quality control involves incubating the enzymes or reagents in reconstituted recombination assays to eliminate the possibility of relevant protein or nucleic acid contaminants that may interfere with recombination assays and lead to misinterpretation of experimental data. Here batch by batch quality controls are done for each new product before it is released to the market. 

This protocol describes the steps in carrying out Nuclease test for:

OpenVent Enzyme
PCR Master Mixes - All formulations (Wet and Dry)
6x DNA loading dye

[BEFORE_START]
Clean and disinfect all work surfaces with a 1:10 dilution of bleach followed by 70% alcohol.
Make sure all resources needed are prepared.

[GUIDELINES]
Before carrying out quality control on products research should be carried out on the possible assays that apply for that particular product and availability of resources.
This protocol describes how to check Nuclease activity for PCR Enzymes, PCR Master Mix and DNA loading dye.

[STEPS]
SECTION: Nuclease activity
1. 
 

SECTION: Nuclease activity
2. Pipetting

Pipette the following reagents into 0.2ml reaction tubes as shown in the table below, while working on ice

 


Incubate reaction tubes for the specified times and briefly place tubes on ice to stop the reaction.

Checking and interpreting results:

After incubation period the reaction is stopped, and a1.5% agarose gel prepared, run and visualized as in this protocol.

Any change in band intensity and migration position of the plasmid DNA bands compared to the negative control signals potential endonuclease activity. Linear DNA should run true to size(known size of plasmid used), supercoiled DNA should run faster and relaxed circle DNA should run slower(a single strand cut).
TITLE: Mimulus in planta Transformation
AUTHORS: Yaowu Yuan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection consists of three protocols: </div><div class = "text-block">1. Agro Transformation </div><div class = "text-block">    a) Using Electropotator </div><div class = "text-block">    b) Using Freeze-Thaw Method</div><div class = "text-block">2. Agro Preparation </div><div class = "text-block">3. Plant infiltration </div></div>

[STEPS]