eamag/protocols-with-steps huggingface dataset:
TITLE: P4. PROCEDIMIENTO DE ADMISIÓN Y MATRICULACIÓN
AUTHORS: cgarcia

[DESCRIPTION]
P4. PROCEDIMIENTO DE ADMISIÓN Y MATRICULACIÓN

[STEPS]
SECTION: P4. PROCEDIMIENTO DE ADMISIÓN Y MATRICULACIÓN
1. Admisión

La Comisión Académica de cada Programa trasladará a la EIDUCAM la resolución de las admisiones.
Secretaría de EIDUCAM comunicará a los doctorandos su admisión o no en el programa, así como los nombres de su tutor y director/es. Esta secretaría también comunicará a cada tutor y director su nombramiento.
En caso de tratarse de la resolución de admisión de la primera fase de admisión La Secretaría de la EIDUCAM, generara la posibilidad de reserva de plaza.
En el mes de octubre se Formalizarán manualmente por parte de la Secretaría de la EIDUCAM en la aplicación LAUREA ACADEMIC, las matriculas de todos los alumnos admitidos 1ª y 2ª Fase, procediendo al envío de las instrucciones de matricula y la Guía de inicio del Programa de Doctorado. Asimismo se procederá a crear en LAUREA ACADEMIC la Ficha de seguimiento de Tesis doctoral y registro de tutor y directores.
SECTION: P4. PROCEDIMIENTO DE ADMISIÓN Y MATRICULACIÓN
2. Primera matrícula:

Una vez admitido en un programa de doctorado de la EIDUCAM, y habiendo recibido la documentación relativa a la formalización de matrícula y guía de inicio, el alumno deberá:

1. Abonar las tasas indicadas en el plazo de 30 días naturales. El no abono de las tasas conllevará que no será efectiva su admisión.
2. Contactar con el tutor asignado
3. Contactar con el director asignado (en su caso)

Además, los estudiantes admitidos con titulación de acceso pendiente de finalizar, deberán justificar, la superación de los títulos de acceso al doctorado. Tendrán de plazo para ello hasta que finalice el plazo de formalización de matrícula.

Transcurrido el periodo de formalización de matrícula, la Secretaría Técnica de la EIDUCAM asignará a los alumnos con matrícula efectiva a su grupo correspondiente para habilitarle todos los recursos que la UCAM pone a su disposición:

-Carnet universitario
- Acceso a biblioteca
- Acceso a recursos digitales
- Campus virtual
SECTION: P4. PROCEDIMIENTO DE ADMISIÓN Y MATRICULACIÓN
3. Renovación de matrícula de tutela académica:

La renovación anual de la tutela académica de tesis doctoral se realizará automáticamente en el mes de septiembre-octubre, teniendo en cuenta la el informe de la Comisión Académica del programa sobre la evaluación anual de cada alumno.

Para ello:

1. La secretaría de la EIDUCAM emitirá un informe de los alumnos que deben renovar su tutela, así como las bajas que se deben efectuar.
2. La Secretaría de la EIDUCAM generará y enviará al alumno las instrucciones para la formalización de su matrícula.
3. El alumno abonará el importe en un plazo de 30 días. El no abono de las tasas conllevará que no será efectiva su renovación.
4. Secretaría Técnica de la EIDUCAM asignará grupo al alumno.
TITLE: Dissection and immunohistochemistry of mouse lung
AUTHORS: Seol-Hee Kim, Thomas Taylor-Clark

[DESCRIPTION]
Mice are euthanized, perfused with fixative for lung tissue collection. Mouse lung is cryosectioned and slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins are visualized using microscopy.

[STEPS]
SECTION: Tissue collection
1. §6 to 8 weeks old mice (male) were euthanized by CO2 inhalation.
SECTION: Tissue collection
2. §Mice were transcardially perfused with phosphate buffered saline (PBS) to remove blood followed by 3.7% formaldehyde (PFA).
SECTION: Tissue collection
3. §Larynx, Trachea and lung were collected. 
SECTION: Tissue collection
4. §Tissue was post-fixed with 3.7% PFA for overnight at 4 °C with gentle agitation.
12. §Tissue slices were blocked with blocking buffer (1% bovine serum albumin/10% normal donkey serum/0.3% PBSTx) for 1.5 h at room temperature.
SECTION: Cryosection
5. §Tissue was washed with ice-cold PBS three times for 30 min. 
SECTION: Cryosection
6. §Tissue was transferred to 30% sucrose solution for cryoprotection and incubated at 4 °C with gentle agitation until it sinks to the bottom of the tube. 
11. §Tissue slices were permeabilized with 0.3 % PBSTx (PBS with 0.3% triton x-100) for 20 min.


SECTION: Cryosection
7. §Lung was flushed 3 to 4 times with PBS containing mL syringe attached with a 23-guage blunt needle, followed by inflating the lung with 2% low melting agarose solution. 
SECTION: Cryosection
8. §After the agarose solution become solidified, the lung lobes were separated and mounted with OCT for cryoprotection prior to the snap frozen in dry ice.
SECTION: Cryosection
9. §Tissue was cut at 80 µm thickness, and the slices were collected in cryoprotectant filled well-plates and stored in -20 °C for further use. 
SECTION: Immunohistochemistry
10. §Tissue slices were washed with PBS three times for 10 min.
13. §Tissue slices were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C.
14. §Tissue slices were washed with 0.2% tween20 in PBS (PBST) for 20 min for three times at room temperature.
15. §Secondary antibodies were diluted in the 1% bovine serum albumin/5% normal donkey serum/0.2% PBST and tissue were incubated with it for 2 h at room temperature.
16. §Tissue slices were washed with 0.2% tween20 in PBS (PBST) for 20 min for three times at room temperature.
17. §Rinse tissue slices with PBS three times to remove detergent and cover-slip with VECTASHIELD® Antifade Mounting Medium with DAPI.
TITLE: Growing Soybean in the Greenhouse
AUTHORS: Lynn  Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for planting, germinating, and growing soybeans in the UIUC RIPE or ACES Plant Care Facility greenhouse.  Has been verified on Petite Havana and Samson cultivars.  </div></div>

[STEPS]
?. Fill classic 1000 pots to within a half inch of the brim with soil.  Tamp the soil down firmly with your hands.
?. Water the soil in thoroughly.  The soybean soil mixes contain high levels of sand and drainage is slow.  It may take several hours for the soil to be saturated and to drain any standing water.
The soil could be saturated and allowed to drain the night before planting.
?. Lay the soybean seed on top of the soil. Push it a few centimeters deep into the damp soil, just enough to be able to cover it with soil.  Do not plant the seed more than a few centimeters deep.
Typical planting depth in the field for soybean is 1-1.5".  That is too deep for the Vodkin or Soybean soil mixes in the greenhouse.  If the soybean is planted more than a few centimeters deep in the greenhouse, it is likely to rot prior to germination.
?. Cover the seed with soil and gently tamp the soil down with your finger.
?. If the soil felt dry at the seed planting depth, water thoroughly.
?. Keep seeds moist at the level of planting but do not overwater.  A light watering every other day is usually sufficient until germination.  Watering can be increased post-germination.
Do not overwater.  Soybean seeds will rot in the soil if planted too deeply or overwatered.
?. See "Guidelines & Warnings" section for plant care guidelines following soybean germination.
TITLE: Statistical analysis
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Statistical analysis</div></div>

[STEPS]
?. Perform statistical analysis with GraphPad Prism 9.
?. Analyze the data of bead-based binding assay  by one-way ANOVA with Dunn’s multiple comparisons test.
?. Analyze the data of ULK1 kinase activity assay  by two-way ANOVA with Šídák's multiple comparisons test.
TITLE: Electroporation of Cas9 protein into human pluripotent stem cells
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
This protocol describes the electroporation of Cas9 protein into human pluripotent stem cells.

[BEFORE_START]
Use ThermoFisher Kit to directly electroporate ESCs with Cas9 protein and sgRNA.
Works better than plasmid transfection.

[STEPS]
1. Add 10 µL to a sterile 1.5 ml tube. Add 6 µg. Then add 1.2 µg. Pipet up and down to mix. Let it sit at Room temperature for 10 min. This is enough for 2 transfections (== one 6 well).

2. While waiting for the Cas9 to bind to sgRNA, individualize cells with Accutase. Neutralize Accutase with 5x volume E8 with Rock inhibitor.
3. Count cells. You will need 2x105 for each transfection.
4. Spin down cells. Let it sit for a while so all the residue media can go down to the bottom of the tube. If the residue media is too much, take it out with a P200 pipet.
5. Resuspend cells to a concentration of 2x105 per 5 μl (ie 4x107 per ml) using buffer R. 
 

6. Prepare a 24 well matrigel coated plate. Add 0.5 mL-1 mL to the wells you will use. Add HAS (1:2500) to each well. Each transfection goes into one well.
7. Wipe the Neon pipet station with EtOH and place it inside the hood.
8. Add 3 mL to the neon tube. Place the tube inside the station. You should feel a click before the tube is securely seated in the station.
9. Use program 13 from the optimization tab for electroporation parameter. Program 9 should also work.
10. When everything is ready, mix 10 µL-11 µL with the Cas9+RNA containing R buffer. The final volume should be in the range of 21 µL-22 µL.
11. Take up a neon tip, pipet 10 µL and electroporate with program 13. 
 


12. If you see air bubble in the tip, take it out, push everything out of the tip and repipet the mixture.
13. If you see sparking during the electroporation, your efficiency will reduce significantly.
14. Once electroporation is complete, push everything into one well of a 24 well plate. Do not pipet up and down with Neon tip.
15. Repeat the same procedure with the same tip and the left over cell mixture. This is just a replicate.
16. Disperse cells evenly in the well and place cells in a low O2 incubator.
17. Put electroporated cells into low oxygen incubator for 2 days (Rm 329, key can be found at Melissa’s desk).
TITLE: L-Type Ca2+ Current Protocol
AUTHORS: Robert  Harvey, Shailesh Agarwal

[DESCRIPTION]
This protocol is for patch-clamp recordings of L-type Ca2+ current and slow delayed rectifier K+ current responses to norepinephrine and acetylcholine.

[STEPS]
1. Membrane currents were recorded from isolated rat venticular myocytes using the whole-cell configuration of the patch clamp technique.
2. Isolated cells were placed in a perfusion chamber (Warner Instruments) on the stage of an inverted microscope (Olympus IX71) and bathed in a K+-free extracellular solution containing (in mM) NaCl 137, CsCl 5.4, MgCl2 0.5, CaCl21.0, NaH2PO4 0.33, glucose 5.5, and HEPES 5 (pH 7.4), maintained at room temperature.
3. Cells were patched using microelectrodes with resistances between 1 and 2 MΩ. Access resistance was compensated to obtain series resistance errors of less than 5 mV.
4. Cells were dialyzed with a K+-free microelectrode solution containing (in mM): CsCl 130, TEA-Cl 20, EGTA 5, MgATP 5, TrisGTP 0.06, and HEPES 5 (pH 7.2).
5. Whole cell currents were recorded under voltage-clamp conditions using a Multiclamp 700B voltage clamp amplifier, Digidata 1440A computer interface, and pClamp 11 data acquisition and analysis software (Molecular Devices). Data were lowpass filtered at 4 kHz, and sampled at 10 kHz.
6. The membrane potential was held at -80 mV. A 50 ms pre-pulse to -40 mV was used to inactivate Na+channels. This was followed by a 100 ms test pulse to 0 mV to elicit the L‑type Ca2+ current.
7. The time course of changes in the amplitude of the Ca2+ current was monitored by recording the amplitude of the peak inward current elicited during the test pulse to 0 mV applied once every 5 s. 
8. A stable baseline (about 5 minutes) was obtained before application of test drugs.
TITLE: Plasmid transduction using competent cell
AUTHORS: An.Huang

[DESCRIPTION]
Plasmid can be transduced into bacteria at competent state using heat shock. This protocol helps transduce plasmid into competent cells.

[STEPS]
2. When the cells are completely thawed, pipette 2 µL plasmid DNA solution into 100 µL competent cell.
Put the cell in ice for 30 min

3. Conduct heat shock on the competent cell by placing the cell in 42 °C water bath for 90 s.
Put the cells back into ice for 2 min

1. Take competent cell out from -80℃ fridge and thaw on ice.
4. Add 900 µL LB broth medium into competent cell mixture. Shake at   for 45 min

5. Centrifuge at 6000 rpm, 5 min.
 

6. Discard 900 µL supernatant and resuspend the pellet in the rest 100 µL supernatant.

7. Spread the cells onto LB agar plates.
 

8. Place the plate with lid on upside for 60 min.

9. Invert the plate and culture at 37 °C in a biomedical incubator overnight.
TITLE: Phage stock preparation
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for phage lambda and phage T7 phage stock preparation</div></div>

[STEPS]
?. Grow E. coli bacterial host (for example LE392, DH10B or DH5alpha) in LB medium overnight at
37 °C
?. Prepare and autoclave  CaCl2 and MgCl2
?. Add of  CaCl2 and  of  MgCl2 to  of LB medium. Make aliquots of 10 mL and inoculate with 0.1 volumes of overnight bacterial host.
50 µl
50 µl
50 ml
?. Incubate with agitation for  at .
37 °C
?. Add  of high titer phage lysate (> 108 PFU/ml).
100 µl
?. Incubate at  with agitation for ∼ or until lysate clears.
37 °C
?. Collect phage lysate and store at until clean up.
4 °C
?. Pool phage lysate into sterile falcon centrifuge tubes and centrifuge at 4,000 rpm for .
50 ml
?. Filter-sterilize the phage supernatant using a 0.22 μm filter to yield a bacterial cell-free phage lysate.
?. Determine phage titer using plaque or spot assay.
TITLE:  Operative Wound Care in Total Knee Artroplasty
AUTHORS: Luisa Mululo, Fabricio Loures, Marcia Vanzillota, José Mauricio Moraes do Carmo, Marcelo Campos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Guidelines for surgical wound care for patients undergoing total knee arthroplasty surgery performed at Hospital Universitario Pedro Ernesto. </div></div>

[STEPS]
TITLE: MTT assay
AUTHORS: Alexander Röntgen

[DESCRIPTION]
Protocol for MTT cytotoxicity assay on SH-SY5Y cells.

[STEPS]
SECTION: General
1. Culture human SH-SY5Y cells at 37 °C and 5% CO2
SECTION: Day 1
3. Count cells and seed 10000 cells/well
SECTION: Day 1
4. Incubate at 37 °C and 5% CO2 for 24 h
SECTION: Day 2
5. Aspirate medium from cells
SECTION: Day 2
6. Add in protein solutions
SECTION: Day 2
7. Place back at 37 °C and 5% CO2 for 24 h
SECTION: Day 3
8. Dilute MTT 1:10 in RPMI medium
SECTION: Day 3
9. Aspirate medium from cells
10. Add 100 µL MTT/RPMI solution per well
11. Place back at 37 °C and 5% CO2 for 4 h
12. Aspirate medium from cells
13. Add 100 µL stop solution per well
14. Incubate at 37 °C, 500 rpm on plate shaker
15. Read out absorbance at 570 nm on plate reader
SECTION: Day 1
2. Detach 80–90% confluent cells with Trypsin-EDTA
TITLE: 4-methylumbelliferyl heptanoate (MUH) - Cell Viability Assay
AUTHORS: Peter Vangheluwe, Shaun Martin, Mujahid Azfar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The 4-methylumbelliferyl heptanoate (</span><span style = "font-style:italic;">MUH</span><span>) assay is a A fluorimetric assay used to study cell-mediated cytotoxicity. The principle behind this assay employs the activity of esterases found in viable cells. These esterases then hydolyse the fluorochrome in the MUH reagent which can then be read using a plate reader.</span></div></div>

[STEPS]
TITLE: Microbial Metabolite Profiling
AUTHORS: contact.microbialtec  

[DESCRIPTION]
Microorganisms and in particular actinomycetes and microfungi are known to produce a vast number of bioactive secondary metabolites. Natural product research needs to continually improve the efficiency of the selection, screening, dereplication, isolation, and structure elucidation processes, in order for natural product chemistry to continue to be competitive with purely synthetic-based discovery methods. For industrially important fungal genera such asPenicilliumandAspergillus, the production of these compounds has been demonstrated to be very consistent at the species level. This means that direct metabolite profiling techniques such as direct injection mass spectrometry or NMR can easily be used for metabolomics of strains from both culture collections and natural samples using modern informatics tools. Metabolomics can be used for the identification and classification of filamentous fungi and for the discovery of novel compounds when used in combination with modern methods for dereplication. Such approaches will be important for future effective drug discovery strategies, especially for the dereplication of culture collections in order to avoid redundancy in the selection of species. This will maximize the chemical diversity of the microbial natural product libraries that can be generated from fungal collections.

[STEPS]
TITLE: SAVE imaging of protein aggregates in cerebrospinal fluid
AUTHORS: mathew.horrocks

[DESCRIPTION]
This protocol describe the procedures used to clean a coverglass surface and perform ThT detection of protein aggregates in CSF (SAVE imaging) on a TIRF microscope. 

[STEPS]
SECTION: Preparation of ThT solution
12. Add 70 uL of PLL to each chamber and incubate for 30 minutes.
SECTION: Preparation of ThT
1. Prepare approx. 4 mM stock of ThT in 100% ethanol and vortex extensively (approx. 1 hour).
SECTION: Preparation of ThT
2. Prepare approx. 200 uM ThT dilution in PBS, vortex thoroughly (approx. 20 mins) and filter through 0.02-micron filter.
SECTION: Preparation of ThT
3. Measure concentration of ThT preparation using a DeNovix spectrophotometer (extinction coefficient 36,000 M-1 cm-1 at 412 nm).
SECTION: Preparation of ThT
4. Prepare a 50 uM working stock in 0.02-micron filtered PBS
SECTION: Addition of sample for imaging
5. Add 5 uL CSF, 5 uL ThT working stock, and 40 uL PBS to an eppendorf tube. 
SECTION: Addition of sample for imaging
6. Add 50 uL of the sample prepared in the previous step to the PLL-treated coverslips, and incubate for 1h. 
SECTION: Addition of sample for imaging
7. Rinse with PBS. 
SECTION: Addition of sample for imaging
8. Add 100 uL of 5 uM ThT to the coverslip. 
SECTION: Imaging
9. Acquire an 8 x 8 grid of 200-micron spaced fields of view per well by total internal reflection fluorescence microscopy using the ONI Nanoimager with 100x/1.4 oil immersion objective lens. Samples are excited at 405 nm, 50 frames captured per field in each channel at 20 frames s-1.
TITLE:  SOP: cell culture methods  for non-model organisms
AUTHORS: Ana Riesgo, María Conejero, C.DiNizo, Jonas Astrin, Cira Martínez, Tomàs Marqués

[DESCRIPTION]
Acquisition of somatic cells from vertebrates for cell cultures is well stablished in the literature. However, some vertebrate taxa like amphibians prove to be challenging mostly because there is a higher chance of contamination in this group or because the cells do not survive for long periods preventing to be cryopreserved. For invertebrates, the challenge is even greater and so far, the protocols tested have not proven satisfactory and are still being developed. Therefore, the SOPs for invertebrates presented below are still in the testing phase.

[STEPS]
TITLE: OG1RF_genomic_DNA
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">OG1RF genomic DNA isolation</div></div>

[STEPS]
?. [Steps]
Generate an overnight culture in 10 mL of BHI
?. [Steps]
Spin down culture in morning for 10 min at 3500 RPM and pour off supernatant.
?. [Steps]
Make 50 mM EDTA (500 µL 0.5M EDTA stock in 4.5 mL water).
?. [Steps]
Add 960 µL of 50 mM EDTA to OG1RF pellet, then aliquot 480 µL of 50 mM EDTA/cells to a 1.5 mL Eppendorf tube.
?. [Steps]
Add 120 µL of 10 mg/mL lysozyme
?. [Steps]
Incubate at 37°C for 60 minutes
?. [Steps]
Spin at 13000 RPM for 2 minutes and pour off supernatant.
?. [Steps]
Add 600 µL nuclei lysis solution
?. [Steps]
Incubate at 80°C for 5 min then cool to room temperature
?. [Steps]
Add 3 µL of RNAse solution
?. [Steps]
Incubate at 37°C for 60 minutes
?. [Steps]
Add 200 µL of protein precipitation solution. Vortex vigorously for 20 seconds
?. [Steps]
Put on ice for 5 minutes
?. [Steps]
Spin at 13000 RPM for 3 minutes
?. [Steps]
Transfer supernatant to a clean Eppendorf tube containing 600 µL of isopropanol
?. [Steps]
Gently invert
?. [Steps]
Spin at 13000 RPM for 2 minutes and pour off supernatant. Be careful to not disturb pellet.
?. [Steps]
Add 600 µL of 70% ethanol to wash DNA pellet. Stream ethanol directly onto pellet without touching the pipette tip to the pellet.
?. [Steps]
Spin at 13000 RPM for 2 minutes and pour off supernatant carefully.
?. [Steps]
Invert and air dry
?. [Steps]
Add 100 µL of DNA hydration solution
?. [Steps]
Incubate at 65°C for 1 hour or leave at 4°C overnight
TITLE: SEM Sample Prep
AUTHORS: Tim D'Angelo, Beth Orcutt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The SEM can be used to directly image biofilms on solid surfaces such as polished rock chips or collected crustal samples. Seawater or fluid bacteria can be imaged after filtering the sample onto a 0.2 μM filter. </div></div>

[STEPS]
?. [Fixation of Sample]
Using sterile forceps, submerge sample in an appropriate amount of 4°C 4% PFA in PBS in a 1.5 ml eppitube
?. [Fixation of Sample]
Incubate in a 4°C refrigerator for 1 - 3 hours.
?. [Fixation of Sample]
Rinse briefly in 1xPBS
?. [Fixation of Sample]
Dispose of PFA waste in the G2 waste container in the Biological Safety Cabinet
?. [Fixation of Sample]
Proceed to HMDS dehydration
?. [HMDS Dehydration]
Fill an appropriate amount of eppitubes or petri dishes with enough of each ethanol solution to submerge the samples
?. [HMDS Dehydration]
Incubate samples in each dilution for 10 minutes, transfer samples between dilutions with sterile forceps. Let ethanol waste evaporate in the hood or dump down the sink while running the faucet.
?. [HMDS Dehydration]
Fill another set of eppitubes or petri dishes with 100% HMDS and incubate samples for 10 minutes.
?. [HMDS Dehydration]
Remove samples and place them in a sterile petri dish to air-dry for 48 hours in the fume hood.  After this period, they will be ready for gold-sputtering and SEM analysis.
?. [HMDS Dehydration]
Dispose of HMDS waste in Q22 waste container in the flammable’s cabinet under the Fume hood in A211
?. [HMDS Dehydration]
Make a dilution series of ethanol in milliq water at the following concentrations; 50%, 60%, 70%, 80%, 90% and 100% ethanol by diluting 100% ethanol in milliq water.Filter all dilutions with a syringe and 0.2uM filter
?. [Mounting]
Wear gloves while mounting.
?. [Mounting]
Label the underside of the aluminum stub with the sample number in sharpie marker.
?. [Mounting]
Peel the carbon disc (two-sided tape) away from the backing and secure to the aluminum stub.
?. [Mounting]
Cut a small strip of conductive tape about 10-12mm long and cut in half lengthwise with a sharp pair of scissors. Peel off the sticky backing and attach to two opposite edges of the two-sided tape.  Press the tape firmly down with forceps to ensure good contact. (Skip until step 6 if you are mounting filters).
?. [Mounting]
Using sterilized forceps, pick up the sample (rock chip etc.) and gently press onto the two-sided carbon tape.
?. [Mounting]
If you are mounting filtered samples, affix the 0.2 μM filter to the two-sided tape, then repeat step 4.
?. [Mounting]
Store mounted samples in the stub holder, preferably in a dessicator or a closed container with a dessicant pack. Keep the filter remains and store likewise.