eamag/protocols-with-steps huggingface dataset:
TITLE: Soluble and insoluble A-SYN fractionation
AUTHORS: michela.deleidi, Hariam Raji, Federico   Bertoli

[DESCRIPTION]
Soluble/insoluble alpha-synuclein fractionation is a technique used to separate different forms of the alpha-synuclein protein based on their solubility properties.

[STEPS]
SECTION: Soluble and insoluble A-SYN fractionation
1. Perform extraction and detection of Triton-soluble (T-sol) and Triton-insoluble (T-insol) alpha-synuclein as described in Stojkovska and Mazzulli(2021). 
SECTION: Soluble and insoluble A-SYN fractionation
2. Lyse individual organoids in 1% Triton X-100 extraction buffer supplemented with 1X PIC, 50 millimolar (mM) NaF, 2 millimolar (mM) NA3VO4 and 0.5 millimolar (mM) PMSF. 

 Extraction buffer
      Triton X-100  1%    NaCl  150 mM    glycerol  10%    HEPES pH 7.4  25 mM    EDTA  1 mM    MgCl2  1.5 mM  
 
SECTION: Soluble and insoluble A-SYN fractionation
3. Homogenize samples with a pestle and incubate on a platform shaker in an ice-water slurry for 30 min, followed by three freeze/thaw cycles and ultracentrifugation at 100000 x g, 30 min, 4 °C. 

SECTION: Soluble and insoluble A-SYN fractionation
4. Collect the supernatant (Triton-X Soluble fraction).
SECTION: Soluble and insoluble A-SYN fractionation
5. Wash the remaining pellet in Triton X-100 extraction buffer followed by another ultracentrifugation at 100000 x g. 

SECTION: Soluble and insoluble A-SYN fractionation
6. Resuspend the pellet in 2% SDS buffer containing 50 millimolar (mM) Tris, pH 7.4 and 1X PIC, boil it for 10 min at 100 °C (Triton-X insoluble Fraction) and label the T-insol fraction. 
SECTION: Soluble and insoluble A-SYN fractionation
7. Sonicate Tx-Insoluble samples for 10 min at 30% power, 20C in a cup horn sonicator (Qsonica-Q700), and then boil them again for 10 min at 100 °C. 

SECTION: Soluble and insoluble A-SYN fractionation
8. Ultracentrifuge Tx-Insoluble samples at 100000 x g, 30 min, 21 °C. 
SECTION: Soluble and insoluble A-SYN fractionation
9. Collect the supernatant (SDS-soluble fraction).
SECTION: Soluble and insoluble A-SYN fractionation
10. Detect protein concentrations using a BCA assay and load 30 µg of total protein for each condition.
TITLE: Troubleshooting guide for  DDNS
AUTHORS: Joyce Akello, Alex Shaw, Catherine Troman, Erika Bujaki, Manasi Majumdar, Aine.OToole, c.ansley, Zoe Vance, rachel.colquhoun, Andrew Rambaut, Javier Martin, Nick Grassly

[DESCRIPTION]
This document provides general guidance for troubleshooting problems encountered in the DDNS method. It outlines the factors that might influence the outcome of the DDNS PCR assay run, the common problems encountered, possible causes of the problems, and provides recommendations on corrective action and preventative action. 
This document is primarily developed for DDNS performed on stool samples, but included are other factors to consider for other sample types.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span class="cit-title">This protocols is from:<br /></span><span class="cit-auth cit-auth-type-author">Linlin Yin</span><span class="cit-sep cit-sep-separator">, et al.</span><span class="cit-auth cit-auth-type-author"> (2015) <a href="http://www.genetics.org/content/200/2/431.long" target="_blank">Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs</a>. </span>Genetics <span class="cit-vol">200<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">431</span><span class="cit-sep">-</span><span class="cit-last-page">441</span></span><span class="cit-elocation">; </span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.176917<br /><br />Please see the<a href="http://www.genetics.org/content/200/2/431.long" target="_blank"> full manuscript</a> for additional details.</span>

[GUIDELINES]
<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/4tcz36.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/4scz36.png" data-ofn="Screen Shot 2015-09-07 at 9.53.47 PM.png" /><br />-------------------<br />List of plasmids deposited at Addgene (Deposit 71794)<br /><br /><strong>Plasmid ID Plasmid Name</strong> <br />64237 pME-Cas9 <br />64239 pGGDestTol2LC-1sgRNA <br />64240 pGGDestTol2LC-2sgRNA <br />64241 pGGDestTol2LC-3sgRNA <br />64242 pGGDestTol2LC-4sgRNA <br />64243 pGGDestTol2LC-5sgRNA <br />64245 pU6a:sgRNA#1 <br />64246 pU6a:sgRNA#2 <br />64247 pU6b:sgRNA#3 <br />64248 pU6c:sgRNA#4 <br />64249 pU6d:sgRNA#5 <br />64250 pU6a:sgRNA(tyr)<br />------------------------<br /><strong>Reference</strong>:<br />JAO, L. E., S. R. WENTE and W. CHEN, 2013 <a href="http://www.pnas.org/content/110/34/13904.abstract" target="_blank">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system</a>. Proc Natl Acad Sci U S A 110: 13904- 13909.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Parse Evercode Fixation Protocol v2.1.1 for cells or nuclei -- University of Minnesota TMCs
AUTHORS: Parse Biosciences, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
https://www.parsebiosciences.com/products/evercode-whole-transcriptome

Fixation for Parse Evercode sc/snRNAseq 
Includes details for both cells and nuclei fixation -- However, each require a different product kit from Parse to complete.  
Please check for the most recent version of the protocol before going forward. 

[STEPS]
1.
TITLE: Visualisation of bacteria around roots
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is carried out to observe the growth of the bacterial biofilm along with the roots in the rhizosphere. In the paper by Bennett, R. and Lynch, J (1981), they inoculated the plant with the bacterial strain and analyzed the growth of the bacterial biofilm around roots. </div></div>

[STEPS]
?. [Removal of roots and bacterial counts]
Carefully remove the roots from the soil.
?. [Removal of roots and bacterial counts]
Divide the roots into three portions: a) 0 to 2 cm from the tip; b) 2 to 4 cm from the tip; and c) 4 cm from the tip to the seed.
?. [Removal of roots and bacterial counts]
Place each sample in a  Erlenmeyer flask containing  sterile distilled water
10 mL
?. [Removal of roots and bacterial counts]
Shake for  on a wrist-action flask shaker (speed setting 7)
?. [Removal of roots and bacterial counts]
Prepare serial 10-fold dilutions and subsequently prepare surface colony counts on nutrient agar plates.
?. [Microscopy]
There are two ways in which you can observe the roots under the microscope.
?. [Microscopy]
Directly mount roots in  of aqueous phenol/glacial acetic acid ( 15:4, by volume). Using a  microscope observe the roots.
?. [Microscopy]
The other method is where you stain the roots by immersing them for  in  aqueous phenol/  aqueous aniline blue/ glacial acetic acid (15:1:4, by volume). Then carefully rinse and mount in  of aqueous phenol/glacial acetic acid ( 15:4, by volume).  Observe the samples using a bright field microscope.
?. [Removal of roots and bacterial counts]
Root samples will have to be examined before and after washing. This is done to determine: a) the pattern and intensity of microbial colonization; b) whether the washing procedure was effective in removing the bacteria from the root surface.
?. [Biofilm Formation]
Inoculate a plate of Bacillus strain (from ) on a BHI agar plate overnight (~) at  .
-80 °C
37 °C
?. [Biofilm Formation]
Inoculate 1:1,000 of broth culture () into  MSgg media in petri dishes.
12 µl
12 mL
?. [Biofilm Formation]
Inoculate BHI broth from overnight agar plate (one colony) and grow for  with shaking (~200 rpm) at .
5 mL
37 °C
?. [Biofilm Formation]
Let biofilm form (covered with dish lid without shaking) at  (usually 3 and  7 days). Ensure that incubator has a water container to ensure the dishes do not dry out.
37 °C
TITLE: OMS Atlas OCT Spatial Mapping
AUTHORS: Brett    Johnson, Danielle  Galipeau, George  Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections an OCT block, prepares the resulting slides and samples, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer OCT blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing.

[STEPS]
SECTION: Preparation
1. Verify the identity of the OCT block to be cut against written request for sectioning.
SECTION: Preparation
2. Remove OCT block from -80 °C freezer and acclimate to cryostat (-20 °C ) for minimum of 180 min.

SECTION: Preparation
3. Label all slides and cryotubes with a unique BEMS ID and Part#, corresponding to the written request and OCT spatial map (below).
 

SECTION: Preparation
4. Prepare PEN membrane slides by exposing close (~15-20cm) to a UV source for 15 min .

SECTION: Sectioning
5. Affix OCT block to cryostat chuck.
SECTION: Sectioning
6. Orient and face block to get adequate amount of core.
Note: Avoid excessive facing to reduce tissue loss.
SECTION: Sectioning
7. Set cryostat to 5 micron sections.
Note: All sections cut from here on should be sequential. The serial order, adjacency, and consistent orientation of the sections are all important factors. Please note any deviations from the protocol.
SECTION: Sectioning
8. Cut first four sections at 5 microns (Part#1-4) and affix onto appropriately labeled slide according to OCT spatial map (step #3 above).
SECTION: Sectioning
9. Change section thickness to 12 microns.
SECTION: Sectioning
10. Cut one section (Part#5) and place in a cryotube.
Note: This is an intermediate section generated when the Cryostat is switching thicknesses. The actual thickness of this section should be about 7µm.
SECTION: Sectioning
11. Cut two sections (Part#6, 7) and place on appropriate membrane slides.
SECTION: Sectioning
12. Change section thickness to 40 microns.
SECTION: Sectioning
13. Cut 2 sections (Part#8) and place both sections in a single cryotube.
SECTION: Sectioning
14. Place all slides, both cryotubes, and remaining OCT block in -80 °C freezer.
Note: No slides are to be fixed under this protocol.

SECTION: Processing
15. Perform hematoxylin and eosin (H&E) staining on slide labeled Part#2 (see OCT spatial map in step #3 above).
SECTION: Processing
16. Deliver unstained slides (Part#1, 3, 4, 6, 7), cryotubes (Part#5, 8), and remainder OCT block (Part#9) to BioLibrary for distribution.
Note: Keep samples frozen at all times. Store at -80 °C. Transfer/ship on dry ice.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a collection of protocols from:<br /><span class="cit-title"><span class="cit-auth cit-auth-type-author">Paix A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Folkmann A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Rasoloson D</span><span class="cit-sep cit-sep-separator">, </span><span class="cit-sep cit-sep-last-separator">and</span><span class="cit-auth cit-auth-type-author"> Seydoux G (2015) <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR/Cas9 Ribonucleoprotein Complexes</a></span>.</span>  Genetics <span class="cit-elocation">genetics.115.179382</span><span class="cit-sep cit-sep-after-article-pages">; </span><span class="cit-ahead-of-print-date"><span class="cit-sep cit-sep-before-article-ahead-of-print-date">Early online </span>July 17, 2015. <span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.179382<br /><br /></span></span>Please see the <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">full manuscript</a> for detail.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: center;"><img style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" width="100" height="90" data-src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" data-ofn="nehirarma-üst-Tr.png" />  </p>
<p style="text-align: center;">Nehir Biyoteknoloji Ltd. <a href="http://www.nehirbt.com.tr/en/" target="_blank">www.nehirbt.com</a></p>
<p style="text-align: center;"> </p>
<p style="text-align: center;">This protocol explains the usage of PDMS microfluidics with an application example of analysis by microscope. Interpretive pictures are added. </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Catalepsy test (Bar test)
AUTHORS: Chuyu Chen

[DESCRIPTION]
The catalepsy test (bar test) was developed to test motor coordination and motor impairments.

[STEPS]
2. The time during which each mouse maintained the position was recorded up to a maximum of 3 minutes for three trials
1. Mice were gently removed from their home cage by their tail and their forepaws were placed over a horizontal bar, 0.2 cm in diameter and fixed at a height of 4 cm above the working surface. 
3. Average of time from three trials was used for analysis
TITLE: BCA Assay for protein quantification
AUTHORS: Karina Conkrite

[STEPS]
?. Use provided 0.2% BSA for creating a standard curve: Dilute amount necessary for the assay 1:2 using lysis buffer
?. Label tubes (1.5 mL Eppendorf) for standards.  Final Conc ug/mL		Volume 0.1% BSA		Volume buffer  Standard (ug)	                Volume 0.1% BSA (uL)        Volume buffer(uL)          0	                                                0	                                        20         2	                                                2	                                        18         4	                                                4	                                        16         6	                                                6	                                        14         8	                                                8	                                        12         10	                                                10	                                        10         15	                                                15	                                          5        20                                                    20                                             0
?. Label tubes for samples.
?. Add diluted BSA to standard tubes last, before adding working reagent.
?. To each sample and all standards, add 1 mL Working Reagent (comprised of [1 mL Reagent A + 20 uL Reagent B] x number of samples + 10% extra for error). Mix well.
?. Place tubes in 37° C incubator for 30 minutes to react.
?. Cool samples to room temp.
?. Aliquot 200 uL from each tube to a well of a clear, 96-well plate. This can be done in triplicate if desired.
?. Read on BCA program on GloMax plate reader at λ = 562 nm
?. Use values from BSA standards to create standard curve. Can be done in Excel, Statmost, or GraphPrizm. Seeking R2 value as close to 1 as possible. Ensure all your samples are within the standard curve range. If not, need to be redone.
?. Use the resultant y=mx+b equation to quantify the amount of protein in each sample. Dividing by 3 will give you your protein concentration per uL.
?. Keeping all protein on ice throughout, use 3 uL/ sample + 17 uL Lysis Buffer for a total volume of 20 uL.
?. Add appropriate amount of Lysis Buffer to all tubes for the standard curve, following chart above.
TITLE: Mouse Behavior - Open Field and T-Maze
AUTHORS: Robert Edwards, Shweta Jain

[DESCRIPTION]
This protocol describes two behavioral tasks for mice. The first is the Open Field Test, which is used to asses motor behavior, and the second is the T-Maze, which is used to assess spatial learning.

[STEPS]
SECTION: Open Field Behavior
1. On each behavior day, bring mice in their home cages to the behavioral area. Blind experimenter to all test variables (e.g. genotype, drug condition, etc.).
SECTION: Open Field Behavior
2. On days 1 and 2, habituate mice to an open field chamber (clear acrylic cylinder, 25 cm diameter) by placing them in the chamber for 30 minutes undisturbed

 

SECTION: Open Field Behavior
3. On day 3, perform test session by placing mice in chamber for 30 minutes and recording their location using an overhead mounted camera
SECTION: Open Field Behavior
4. Calculate average movement velocity and total distance traveled using commercial video tracking software (Noldus EthoVision)
SECTION: T-Maze
5. Prior to T-Maze task, handle mice every day for one week and calorie restrict to reduce weight to ~85% of original body weight; measure weight daily.
SECTION: T-Maze
6. For two days before the task
familiarize mice with the food reward by providing 10 reward pellets in their home cage
familiarize mice the T-maze by allowing each mouse to explore the maze freely for 10 minutes with 5 rewards in each arm
SECTION: T-Maze
7. Simple uncued task (food provided in the left arm of the T-maze); repeat for 10 trials on each test day
SECTION: T-Maze
7.1. Place mouse in the start area for 60 seconds
SECTION: T-Maze
7.2. Remove sliding door, give mice 60 seconds to choose the left or right arm; if choice is not made within 60 seconds, nudge into either the left or right arm on an alternating basis
SECTION: T-Maze
7.3. Place door in front of the entered arm, give mice 60 seconds to consume the reward pellet or remain in the non-reward arm
SECTION: T-Maze
8. Uncued task
add a mold over the arms of the T-maze with vertical stripes for the reward side and horizontal stripes for the non-reward side
the striped visual cue is assigned pseudo-randomly to the left and right arms, maintaining a 50:50 ratio overall
Task is repeated for 20 trials each test day, except in β3B KO experiments (in Jain et al., 2023) where 14 trials were performed on days 1-4 and 20 trials were performed on subsequent days
SECTION: T-Maze
8.1. Place mold with striped visual cues on maze; add food reward pellets to the vertical striped side
SECTION: T-Maze
8.2. Place mouse in the start area for 60 seconds
SECTION: T-Maze
8.3. Remove sliding door, give mice 60 seconds to choose the left or right arm; if choice is not made within 60 seconds, nudge into either the left or right arm on an alternating basis
SECTION: T-Maze
8.4. Place door in front of the entered arm, give mice 60 seconds to consume the reward pellet or remain in the non-reward arm
TITLE: Loading Syringes to Inject into Microfluidics Device
AUTHORS: freeman.lan

[DESCRIPTION]
Protocol for loading syringes with small amounts of liquid sample for injection into droplet microfluidic devices. 

[STEPS]
SECTION: Preparing Syringes
1. Take out a syringe 
 
Add 27g needle to the syringe
 

SECTION: Preparing Syringes
2. Use the needle and syringe to draw out approximately 200 µL of   
SECTION: Preparing Syringes
3. Cut approximately 20 centimeters of   with a razor and attach it to the needle tip. 
 


SECTION: Loading the Syringe
4. Push the syringe so that the HFE7500 oil starts dripping at end of the tubing.

 

5. Put the tube into the solution that you would like to load, and slowly draw the syringe to pull the solution into the tubing and syringe.
 

SECTION: Interfacing with the Device
6. Attach the loaded syringes to the syringe pumps 
 

 

7. Insert the other end of the tubing into the appropriate hole in the microfluidic device, using a tweezer or by hand.
8. Set the appropriate syringe diameter and pump rates into the syringe pump and press start. You may want to begin the pumps with a higher pump rate in order to get the syringes to pressurize more quickly.
TITLE: RNA Integrity Check
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA Integrity Check: Modification of Protocol from Qiagen</div></div>

[STEPS]
?. [RNA Integrity Check]
Determine the concentration of RNA; ideally want to run approximately   of RNA on the gel (should be about   of your sample)
1 µg
1 µl
?. [RNA Integrity Check]
Spray gel tray, comb, damns, and a small flask with RNAse Zap.
?. [RNA Integrity Check]
For small gels, melt 0.3 grams of RNA ONLY agarose in 30 ml 1X TBE (or 1X TAE) to the cleaned flask (for a 1% gel using Horizon gel boxes.  If using something else, adjust accordingly).
?. [RNA Integrity Check]
Let the solution cool slightly, add   .  Pour gel and let solidify.
[EtBr]
?. [RNA Integrity Check]
For samples:  RNA sample in a total of   of water (if RNA concentration is about  , add   of RNA to   of RNAse-free water)  of sample bufferSample Buffer (make FRESH!)  (80% glycerol, 0.2% bromophenol blue, 0.2% xylene cyanol; this does NOT need to be made fresh!)(1/10  of the recipe above is normally enough)
8 µl
[/ml]
1 µl
7 µl
4 µl
[of concentrated dye]
[of formamide]
[of 37% formaldehyde]
?. [RNA Integrity Check]
Load gel and include a 1 kb DNA ladder for size approximation; run at 100 volts about 45-60 minutes
?. [RNA Integrity Check]
Gel: should see 2 distinct bands corresponding to the 16s and 23s rRNA species.  If RNA is intact, will not see smearing. Should also see lots of tRNA at the bottom.
Formamide and formaldehyde are toxic and dangerous chemicals.  Handle with care in the hood!
On occasion, I have run a mini-1X MOPS gel to validate RNA integrity especially if I do not have much sample left and really needed to use the RNA for an important experiment.
TITLE: Reto F1016B.201.01
AUTHORS: Natalia Gámez, Diego  Zazueta Acosta

[DESCRIPTION]
-Armar un circuito RC
-Medir el tiempo de carga del capacitor
-Obtener mediciones e importarlas a matlab para compararlas con nuestro modelo matematico simulado 


[STEPS]
SECTION: Traducir el valor de las resistencias
1. Se necesita traducir los Ohms (Ω) que nos dan cierta combinacion de colores en las resistencias para así saber cual debemos utilizar. 
Puedes encontrar una imagen en internet que te diga cuanto vale cada banda de color, en nuestro caso utilizamos el siguiente sitio web:
https://www.digikey.com.mx/es/resources/conversion-calculators/conversion-calculator-resistor-color-code

  

 
  

SECTION: Montar el circuito
2. Para esta parte del experimento se necesitaran los siguientes materiales:
1 Capacitor de 10μF
1 Capacitor de 100μF
Resistencia de 1000 Ohms 
2 Resistencias de 100 Ohms 
1 Protoboard 
Cables caiman 

Procedemos a montar el circuito para posteriormente concertarlo a la fuente de poder y al osciloscopio.
  

SECTION: Como montar el circuito
3.  Colocas el primer cable en la mitad del protoboard, paralelo a este cable se coloca el segundo cable en la fila negativa del protoboard.
Conectas el cable caiman positivo y el segundo cable caiman al negativo.
Colocas la resistencia R1 y R2 paralelas a la resistencia RC.
Ajustas la fuente de energia para comenzar la carga y descarga de los capacitores 
Antes de iniciar el experimento debes de verificar de que el capacitor este totalmente descargado 

SECTION: Calibración de osciloscopio
4. Calibrar el osciloscopio con los limites de 1V y 500ms 

2. 
SECTION: Calibración de osciloscopio
5. Colocar la fuente de energía a 5V
SECTION: Extracción de datos
6. En la pantalla del Osciloscopio deberán de verse tus resultados 
SECTION: Extracción de datos
7. Tomas las medidas de voltaje en la carga y descarga 
SECTION: Extracción de datos
8. Guardas los datos en una memoria USB 
SECTION: Extracción de datos
9. Se repite este proceso 3 veces