eamag/protocols-with-steps huggingface dataset:
TITLE: DESS (DMSO/EDTA/NACL) Protocol
AUTHORS: Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell, Jenna Brown

[DESCRIPTION]
DESS (DMSO/EDTA/NACL) protocol instructing how to make the solution for the preservation of samples. 

[STEPS]
SECTION: Make 2L stock solution of 0.5M EDTA; if stock is already made, proceed to step 4.
1. Combine 372.24 g disodium EDTA and 500 mLdeionized water.
Tip: Disodium EDTA is labeled PINK in the Bik Lab.
SECTION: Make 2L stock solution of 0.5M EDTA; if stock is already made, proceed to step 4.
2. Add enough 5M NaOH to the solution to bring it to a pH of pH 8.0
Tip: NaOH is labeled GREEN in the Bik Lab.
Tip: In some cases, this can be as much as 500 mL. The EDTA will begin to dissolve around a pH of pH 7.0.
SECTION: Make 2L stock solution of 0.5M EDTA; if stock is already made, proceed to step 4.
3. Bring final volume to 2 Lwith deionized water once all disodium EDTA has dissolved.
SECTION: Prepare 2L DESS from 0.5M EDTA.
4. Combine 1 L 0.5M disodium EDTA, 400 mL Dimethyl Sulfoxide/DMSO, and 600 mL deionized water in a 4000 mL Erlenmeyer flask.
Tip: 0.5M disodium EDTA is labeled YELLOW and Dimethyl Sulfoxide/DMSO is labeled ORANGE in the Bik Lab.
SECTION: Prepare 2L DESS from 0.5M EDTA.
6. Add enough NaCl to saturate the solution: Add NaCl in 100g increments until 300g is reached, and then proceed to adding smaller amounts until salt is no longer dissolving.  Solution should saturate with the addition of ~300-400g NaCl. 
Tip: NaCl is labeled RED in the Bik Lab. 
Tip: This step can take hours: BE PATIENT. There may be leftover salt residue once the solution is fully saturated.
SECTION: Prepare 2L DESS from 0.5M EDTA.
5. Put magnetic stir bars in the solution and place flask onto a hot plate. Turn the magnetic stirring function on and leave it at room temperature.
TITLE: Make LB agar medium
AUTHORS: Gurdon Institute mediak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Make LB agar medium.</div></div>

[STEPS]
?. 
?. ABCDEF1Solution&nbsp;LB (ready mix) + Agar  2Strength pH:&nbsp;7.0Batch size5L3      45Ingredients  Quantity  6LB mix  125g 7NaOH 10Mfew drops 8Distilled H2Oup to 5L 9Agarper 1L bottle14g 10 per 500ml bottle7g 11      1213Method     141In the fume cupboard, add the LB mix&nbsp; to 2L&nbsp;
  distilled H2O
  in a bell jar152Transfer the bell jar&nbsp; to
  a magnetic stirrer on the bench163Add distilled H2O to approx. 4L mark&nbsp;
  using a jug174Allow the solutes to dissolve&nbsp;
  then adjust pH to 7.0 using 10M NaOH185Adjust final volume to 5L using&nbsp;
  distilled H2O196Aliquot (using a pump), label &amp; date&nbsp;207Dispense 14g&nbsp; agar and a
  magnetic flea to each 1L bottle (OR 7g agar and a magnetic flea to each 500ml bottle)218Autoclave22
ABCDEF1Solution&nbsp;LB (ready mix) + Agar  2Strength pH:&nbsp;7.0Batch size5L3      45Ingredients  Quantity  6LB mix  125g 7NaOH 10Mfew drops 8Distilled H2Oup to 5L 9Agarper 1L bottle14g 10 per 500ml bottle7g 11      1213Method     141In the fume cupboard, add the LB mix&nbsp; to 2L&nbsp;
  distilled H2O
  in a bell jar152Transfer the bell jar&nbsp; to
  a magnetic stirrer on the bench163Add distilled H2O to approx. 4L mark&nbsp;
  using a jug174Allow the solutes to dissolve&nbsp;
  then adjust pH to 7.0 using 10M NaOH185Adjust final volume to 5L using&nbsp;
  distilled H2O196Aliquot (using a pump), label &amp; date&nbsp;207Dispense 14g&nbsp; agar and a
  magnetic flea to each 1L bottle (OR 7g agar and a magnetic flea to each 500ml bottle)218Autoclave22
TITLE: Differential gene expression analysis
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Differential gene expression analysis from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. Differential gene expression analysis in TRAP samples (including calculation of gene enrichment and depletion, relative to tissue homogenate RNA) was performed using DESeq2 (v1.36.0, RRID:SCR_015687) in R (v4.2.1, RRID:SCR_001905) with Bioconductor (v3.15, RRID:SCR_006442). Adaptive shrinkage of log fold change estimates was performed using ashr. The following settings were changed from defaults: minReplicatesForReplace = Inf, cooksCutoff = Inf, filterFun = ihw, lfcThreshold = log2(1.05). Genes were classed as significantly differentially expressed with an FDR-adjust P value < 0.01. Protein-protein interactions were obtained using STRINGdb (version 11) with a minimum confidence threshold of 0.4.
TITLE: Archived Human Tissue Collection 1999-Aug 2015 -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Liver and adipose tissue samples obtained from the UMN Biospecimen Repository that were collected and stored between 1999 and August 2015 not have documented collection protocols. This protocol includes the relevant information that was recorded at sample collection.  

[STEPS]
1. Adipose and liver samples collected and stored between 1999 - Aug. 2015 do not have a record of tissue collection. 

The following information was documented and is available for each tissue: 
- Date and time of collection
- Processed time 
- Age
- Ethnicity 
- Race
- Gender 
- Material group and type
- Volume
- Vial modifiers
- Pathology diagnosis 
- QC tissue status and diagnosis
TITLE: Vandy – Energy Balance with Promethion
AUTHORS: Louise Lantier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>The Promethion from Sable Systems (Las Vegas, NV) assesses several key metabolic characteristics of mice such as energy expenditure, preferred metabolic substrate, meal patterns, activity, live body mass. These are measured by individually housing the mice in the Promethion System cages for several days during which numerous parameters are continuously measured (food and water intake, weight, activity, O</span><span style = "vertical-align:sub;">²</span><span> and CO</span><span style = "vertical-align:sub;">²</span><span>). </span></div></div>

[STEPS]
?. One week prior to the experiment start date, mice are singly housed for acclimation.
?. On the day of the experiment start date, mice are weighed and body composition is assessed.
?. The mice are placed in the cages of the Promethion system, one mouse per cage. The cages are housedin a light and temperature controlled chamber. Default temperature is 23ºC, but it can be set from 10 to 40ºC to monitor mice in hypothermic of at thermoneutrality conditions. Likewise, the light cycle is seton a 12:12h cycle (6am-6pm) but can be modified to suit to study circadian rhythms.
?. Mice are left undisturbed for 5 days during which all the measurements are made.
?. After 5 days, the mice are removed from the cages, and body composition and weight are measured again.
TITLE: SPARC - Attune NxT Set-up for Milli-metabolic bead assay Acquisition
AUTHORS: J Paul Robinson

[DESCRIPTION]
This protocol is to demonstrate how to run a standard plate-based assay on the flow cytometer to produced appropiate concentations of the various analytes to be measured.

[BEFORE_START]
Setting up instruments correctly is a critical step in any assay

[GUIDELINES]
This assays is specifally designed for the ATTUNE NxT flow cytometer and the Milli-metabolic bead assays

[STEPS]
1. Check the waste and 

If the waste is full, disconnect the tank and dispose of the waste down the sink with running water. Add 200 mL of 5% bleach to the tank and reconnect to the Attune NxT. Fill the Focusing Fluid tank from the focusing fluid cube.

2. Check and fill as needed the shutdown solution reservoir and the wash solution reservoir.
3. Also check the waste and focusing fluid reservoirs at the plate sampler station.
4. At the workstation, click on instrument > start-up at the top of the screen.
5. When start-up is complete, the indicator lights at the instrument will be green.
6. Run the performance test.
7. When the plate finishes, right click on the experiment.
8. Select export FCS files
9. Copy files onto the appropriate directory
10. Analyze data in Mplex software
11. Once data files are analyzed in MPLEX, follow the MPLEX protocol to send data for depositing to NIH.  See this protocol for details:
TITLE: Sample fixation of biopsy tissue for Electron Microscopy (EM)
AUTHORS: Jessica Riesterer, Erin Stempinski, Claudia Lopez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The most crucial step in the entire electron microscopy workflow is sample fixation. Tissue needs to be preserved in strong fixative as soon as possible to maintain cellular ultrastructure. In a clinical setting, fixation time is critical in order to capture precious human tissue adequately. Of course, priority is given to patient care, and therefore, the tissue sometimes cannot be handled as quickly as needed for optimal ultrastructure preservation. However, we recommend 2 minutes as a “best practice” time to start of preservation. </div></div>

[STEPS]
?. In order to facilitate quick fixation, the team of clinical coordinators should be provided with Eppendorf tubes containing 1.5 mL of fixative solution to have on-hand in the operating room during biopsies using 18-gauge core needles where 3-4mm of core is preserved. Larger volumes may be required for resections.
?. Karnovsky’s fixative (2.5% paraformaldehyde, 2.5% glutaraldehyde in 0.1M Na Cacodylate buffer (pH 7.4)) is the solution of choice (Karnovsky, 1965).
?. Eppendorf tubes with and without tissue are stored at .
4 °C
?. Tissue should be placed gently into the Eppendorf tube, ensuring that the tissue is completely submerged in fixative solution to prevent drying out. Use of forceps should be avoided whenever possible to minimize mechanical damage. An orange stick or scalpel blade can be used to "scoop" the tissue into the tube.
TITLE: Extracellular vesicle isolation from bacterial cultures
AUTHORS: Steven Biller

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Steps for isolating extracellular vesicles and other small particles from a bacterial culture</div></div>

[STEPS]
?. [Culturing]
Grow bacterial culture to mid/late exponential phase
?. [Vesicle+Particle Isolation]
Remove cells by filtration through a 0.2µm filter and collect the filtrate (
?. [Vesicle+Particle Isolation]
Concentrate
?. [Vesicle+Particle Isolation]
Re-filter concentrated material through a 0.2 µm syringe filter. Pellet the vesicles in an ultracentrifuge at 100,000 xg, for at least 1 hr, at 10 C or lower.
?. A pellet will not necessarily be visible. Remove as much of the supernatant as possible. If desired, wash the vesicle pellet in the appropriate buffer (media, 1x PBS, etc) as needed for downstream application.
?. [Vesicle+Particle Isolation]
Resuspend final vesicle pellet in buffer. Store at -20 or -80 C.
TITLE: 20 minute PCR Enzymatic Cleanup
AUTHORS: Julian Liber

[DESCRIPTION]
A rapid microplate-compatible protocol for cleanup of PCR products prior to Sanger sequencing. Verify that the PCR produces a single band, and retain some PCR product for cleanup and sequencing. Set up at room temperature, and react on the thermocycler.

Mechanism: PCR products contain 1) primers and 2) dNTPs which will interfere with downstream Sanger sequencing reactions. Exonuclease I degrades ssDNA (primers) and calf intestinal phosphatase degrades dNTPs. Functions like NEB #E1050.




[STEPS]
SECTION: Making the enzyme master mix
1. Add to a 1.5 mL sterile conical tube
760 uL water
94 uL 10x rCutSmart buffer
96 uL 10x Exonuclease I buffer (NEBuffer r3.1)
4 uL Quick CIP (5U/uL) NEB #M0525
10 uL Thermolabile Exonuclease I (20U/uL) NEB #M0568
SECTION: Making the enzyme master mix
2. Mix, then aliquot 200 uL into separate tubes. Store at -20C. Good for at least 2 years.
4.  Seal tubes or plates and place on a thermocycler. React at 37ºC for 15 minutes, then 80ºC for 1 minute.
SECTION: PCR Cleanup
3. Mix 1.2 uL of master mix with 1.5 uL PCR product in PCR strip tubes or plates.
 
 
 

5. Add the product to Sanger sequencing reactions. 1 uL of product, 1 uL of 10 uM primer, and 10 uL of water per tube. If the band is faint, you can use 2 uL or more of product.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the quick version of the Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020). For the full protocol, please click <a href="https://www.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" target="_blank">here</a>.

[BEFORE_START]
Add 4 volumes of ethanol (≥ 95%) to one volume of DNA Wash Buffer. <br />
<ul>
<li>For 50-prep kit, add 20 ml of ethanol to 5 ml of Monarch DNA Wash Buffer</li>
<li>For 250-prep kit, add 100 ml of ethanol to 25 ml of Monarch DNA Wash Buffer</li>
</ul>
All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).<br /><br />Please note: column holds 800 μl

[GUIDELINES]
For detailed protocol and more information, visit <strong><a href="https://www.neb.com/products/t1020-monarch-dna-gel-extraction-kit" target="_blank">www.neb.com/T1020<br /></a></strong><br />The full protocol is available <a href="https://www.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" target="_blank">here</a>. <br /><strong><a href="https://www.neb.com/products/t1020-monarch-dna-gel-extraction-kit" target="_blank"><br /></a></strong><a href="https://www.neb.com/products/t1020-monarch-dna-gel-extraction-kit" target="_blank"></a>A video protocol is available <a href="https://www.neb.com/tools-and-resources/video-library?videoid=%7B9113b074-49b0-4fdf-910e-6426f17c049a%7D" target="_blank">here</a>.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Dissolved silica colorimetric assay using a plate reader (96-well plate)
AUTHORS: Jian Gong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the adaption of a silica colorimetric assay originally described in </span><a href="https://epic.awi.de/id/eprint/39262/1/Strickland-Parsons_1972.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Strickland and Parsons, 1972</span></a><span> (p65-70), modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard </span><a href="https://www.sigmaaldrich.com/catalog/product/SIGMA/CLS9017?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">96-well plates</span></a><span> for rapid measurements of 1 mL, diluted water samples. This assay measures the concentration of molybdate-reactive silica (monomeric and short polymers of silicic acid) in solution.</span></div><div class = "text-block"><span>Samples for this assay should be filtered (</span><a href="https://www.sigmaaldrich.com/catalog/product/aldrich/wha67802502?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">0.2 µm syringe filter</span></a><span>) and placed in </span><a href="https://www.sigmaaldrich.com/catalog/product/sigma/ep022363344?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">2 mL microcentrifuge tubes</span></a><span>. Avoid sample freezing. Store samples in the dark whenever possible.</span></div></div>

[STEPS]
?. At room temperature, in 2 mL microcentrifuge tubes, add rapidly  reagent A (molybdate solution) to of sample/standard/blank solutions while using the pipet (dispensing up and down a few times) to help mix the mixture. Let the mixture stand for  (to allow silicomolybdate complex to form). At this point, if there is enough silica, the solution would turn yellow.
400 µl
1 ml
?. Add  reducing reagent rapidly into each tube, while also mixing a few times with the pipet. The total mixture of each tube should now be 2 mL. The color of the solution should instantly turn from yellow to blue (if there is silica). The intensity of the blue color will increase gradually in time. Allow incubation of , in the dark before absorbance measurement at 810 nm. The system at the Bosak Lab is a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA). Measure by loading 5x replicates of 200 μl mixtures on a 96-well plate.
600 µl
TITLE: BDA Histology
AUTHORS: Blake Butler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Staining protocol for visualization of BDA 3K.</div><div class = "text-block">Species - Feline</div><div class = "text-block">Tissue Preparation - Perfusion of 4% PAF; Cryoprotected 30% Sucrose; Section thickness of 60 microns</div></div>

[STEPS]
?. [Preparatory Steps]
Reconstitute the Vectastain ABC solution Prepare 80 mL solution: Physiological Buffer Solution A Solution BThaw 6 x 1mL vials of NGS for later use
79 ml
480 µl
480 µl
?. [Histological Reaction]
?. [Histological Reaction]
Rinse sections in 0.1 M Physiological Buffer (4x5min)
?. [Histological Reaction]
Block Endogenous Peroxidase  Methanol  De-ionized Water Hydrogen Peroxide
105 ml
45 ml
750 µl
?. [Histological Reaction]
Rinse sections in 0.1 M Physiological Buffer (4x5min)
?. [Histological Reaction]
Transfer tissue to individual reaction wells and incubate in Avidin/Biotin HRP Complex Prepared in Step 1
?. [Histological Reaction]
Transfer tissue back to reaction trays and rinse sections in 0.1 M Physiological Buffer (3x10min)
?. [Histological Reaction]
Incubate in DAB-Nickel Chromogen Solution Physiological Buffer 3,3' Diaminobenzidine Tetrachloride (DAB) Cobalt Chloride (Added dropwise) Nickel Ammonium Sulfate (Added dropwise) Hydrogen Peroxide (Added 60s before incubation)  (Approximate time - Actual endpoint determined visually)
150 ml
0.1125 g
1.875 ml
3 ml
50 µl
?. [Histological Reaction]
Rinse sections in 0.1 M Physiological Buffer (3x5min)
?. Mount from 0.01 M Physiological Buffer
TITLE: Transfection of HT22 cells with Lipofectamine LTX
AUTHORS: Signe Goeke

[DESCRIPTION]
Transfection of HT22 cells

[STEPS]
SECTION: Day 0
1. Plate HT22 cells in 6 well plate: 250K per well
If you want to perform transfection next day plate 500K.


SECTION: Day 1
2. Check cells --> less than ~60-70% confluent wait another day
SECTION: Day 3
3. Dilute the optimized amount of plasmid DNA in 500 μl Opti-MEMI Reduced Serum Medium (no A/A, no FCS). Mix thoroughly.
SECTION: Day 3
4. Mix PLUS™ Reagent gently before use, add 2.5uL volume of PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5 minutes at room temperature.
SECTION: Day 3
5. Mix Lipofectamine LTX gently before use, and add 5uL  directly to the diluted DNA. Mix thoroughly.
SECTION: Day 3
6. Incubate for 30 minutes at room temperature (DNA-lipid complexes are stable for 6 hours at room temperature)
SECTION: Day 3
7. Add ~500 μl DNA-lipid complex (step 4 above) dropwise to the well containing cells. Mix gently by rocking the plate back and forth.
SECTION: Day 3
8. Incubate the cells at 37°C in a CO2 incubator for 18–48 hours prior to testing for transgene expression. 
SECTION: Day 3
6.1. Meanwhile: Add 2mL of antibiotic-free medium containing 5% FCS
SECTION: Day 3
9. Change medium after 4–6 hours to regular medium (with A/A and 10% FCS)
TITLE: Staining and imaging of mouse submandibular ganglion by α-bungarotoxin and nanosensor
AUTHORS: Hongrong Yang, Junfei Xia, James Monaghan, Heather A Clark

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to label acetylcholine receptors in isolated submandibular ganglion from mice using α-bungarotoxin and nanosensor formulated by the Clark lab.</div></div>

[STEPS]
?. [Staining of submandibular ganglion by α-bungarotoxin and nanosensor (formulated by Clark Lab)]
1. Rinse submandibular ganglion with PBS for 2. Soak submandibular ganglion in for .Alternatively, soak submandibular ganglion in nanosensor solution formulated by Clark Lab for 3. Rinse submandibular ganglion with PBS for .
500 µl
500 µl
?. [Imaging of submandibular ganglion]
1. Place the submandibular ganglion on a glass slide and cover it with cover slip.2. Image the whole tissue using a Zeiss LSM 700 confocal. Use 488 nm laser line for GFP and Oregan Green 488 channel, 555 nm laser line for pHAb, and 639 nm laser line for bungarotoxin-Alexa Fluor 647 channel. Set laser power at 2% or at the appropriate power intensity that cellular structures can be clearly seen.
?. [Extraction of submandibular ganglion]
1. Enthanize animals using CO2.2. Make a midline incision in the neck and expose the salivary glands.3. Separate the pair of salivary glands and expose the salivary ducts.4. Remove connective tissues surrounding salivary ducts and cut the ducts from where they enter salivary glands to where they enter digastric muscles. The submandibular ganglion are attached to cutted ducts.
Well isolated salivary ducts with at least one submandibular ganglion attached
TITLE: Reverse-phase high pH fractionation, using the Pierce kit
AUTHORS: Louise Uoselis

[DESCRIPTION]
This protocol uses the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, Cat# 84868)

[STEPS]
SECTION: Conditioning the columns
1.      Remove the white cap on the end of the column and place the column in a 2 mL collection tube
SECTION: Conditioning the columns
2.      Centrifuge at 5000 rcf, 2 min at Room temperature, discard the liquid

SECTION: Conditioning the columns
3.      Remove the screw cap and add 300 µL ACN to the column (replacing the screw cap after)

SECTION: Conditioning the columns
4.      Centrifuge at 5000 rcf, 2 min at Room temperature, discard the liquid

SECTION: Conditioning the columns
5.      Repeat steps 3 and 4 (for a total of 2 washes with ACN)
SECTION: Conditioning the columns
6.      Repeat steps 3 – 5 with 0.1% TFA instead of ACN (total of 2x washes with 0.1% TFA)
SECTION: Conditioning the columns
7.      The column is now ready to use
SECTION: Fractionating the samples
8. Add 300 µL of 0.1% v/v trifluoroacetic acid to each sample

SECTION: Fractionating the samples
9. Vortex for ~ 10 s

SECTION: Fractionating the samples
10. Leave to incubate for 5 min at Room temperature

SECTION: Fractionating the samples
11. Vortex for ~ 10 s

SECTION: Fractionating the samples
12. Sonicate in a waterbath sonicator for 5 min in an ice slurry

SECTION: Fractionating the samples
13. Load each sample into a fractionation column, replace the cap and centrifuge at 3000 rcf, 2 min (keep eluate as ‘flow through’ fraction)

SECTION: Fractionating the samples
14. Place the column into a new tube, and load 300 µL of water, and centrifuge at 3000 rcf, 2 min(keep eluate as ‘wash’ fraction)

SECTION: Fractionating the samples
15. Place the column into a new tube, and load the TMT wash solution (5% ACN, 0.1% TEA) 
SECTION: Fractionating the samples
16. Place the column into a new tube, and load 300 µL of the appropriate elution solution (see table below), and centrifuge at 3000 rcf, 2 minto collect the fraction. 

SECTION: Fractionating the samples
17. Repeat step 5 for each step of the gradient fraction.
SECTION: Fractionating the samples
18. If you are concatenating the fractions, combine the fractions into the desired combinations

SECTION: Fractionating the samples
19. Lyophilise all samples until there are only a few µL left in the tube