eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>ThI high sensitivity miRNA library generation for the Illumina sequencing platform. Our enhanced reagent kit enables the discovery and profiling of small RNAs from a variety of sources including FFPE, exosome, serum, and whole blood. The TailorMix workflow is designed for ease of use, enabling library preparation a single day.</p>
<p> </p>
<h3>Features:</h3>
<ul>
<li><strong>User Friendly Workflow:</strong> ­ Libraries can be prepared in a single day.</li>
<li><strong>No Additional Reagents Necessary:</strong> All reaction enzymes and buffers are provided.</li>
<li><strong>Stress Free Gel Purification:</strong> <a href="http://www.seqmatic.com/products/tailorcut-gel-extraction-tool-set/" target="_blank">TailorCut Gel Extraction Tool Set</a> is included for easy gel excision and purification.</li>
<li><strong>Easy to Use:</strong> Reagents are supplied as ready-to-use mixtures which improves consistency and reproducibility.</li>
<li><strong>Ultra High Sensitivity: </strong>Prepare miRNA libraries from any source with as little as 10ng of total RNA input<strong>.</strong></li>
</ul>

[GUIDELINES]
<p><strong>Figure 1</strong> TailorMix miRNA Sample Preparation V2 Overview</p>
<h1> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dz5effe.png" width="735" height="423" data-src="https://s3.amazonaws.com/pr-journal/dz4effe.png" data-ofn="1.png" /></h1>
<h3> </h3>
<h1>Best Practices</h1>
<p> </p>
<ul>
<li>Always wear gloves and use sterile technique.</li>
<li>Set up reactions using sterile non-stick nuclease-free tubes.</li>
<li>Place samples and reagents on ice or on chilled cooler block at all times and avoid extended pauses.</li>
<li>Reagents should be prepared using RNAse-free components</li>
<li>Prepare an extra 10% mixture when running multiple samples.</li>
<li>Avoid repeated freeze/thaw cycles.</li>
</ul>
<h6> </h6>
<h1>RNA Input</h1>
<p> </p>
<p>This protocol has been optimized using 10 - 100 ng of purified high quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. You may also use isolated miRNA as the starting material.</p>
<h1> </h1>
<h1>Sample Pooling Guidelines</h1>
<p> </p>
<p>The TailorMix MiRNA Sample Preparation kit is capable of multiplexing up to 96 samples into a single lane of an Illumina flow cell. While processing multiple samples in parallel, use a unique index primer for each sample at the PCR step. Samples can be pooled before or after the library purification step.</p>
<p> </p>
<p> </p>
<p> </p>
<h3>Citations &amp; References:</h3>
<p>Niu, Jinzhi, et al. “<a href="http://www.sciencedirect.com/science/article/pii/S0965174815300801" target="_blank">In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.</a>” <em>Insect biochemistry and molecular biology</em> 70 (2016): 127-137.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a slightly modified version of Thermo Fisher's gateway LR protocol which uses less enzyme and is therefore more economical.</p>

[STEPS]
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TITLE: Plasmid Expansion
AUTHORS: Ali Albalakhi, Ning Xia

[DESCRIPTION]
This is the protocol for plasmid expansion.

[STEPS]
1. Thaw the competent cells on ice
2. Chill approximately 5 μg (2 μl) of Plasmid DNA in 1.5 mL microcentrifuge tube
3. Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds*. Do not mix.
6. Add 950 μl of room temperature media* to the tube.
7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Spread 50–100 μl of the cells and ligation mixture onto the plates.
10. Incubate overnight at 37°C.
10.1. Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period,
please follow the recommendations provided by the competent cells’ manufacturer.
SECTION: Plasmids and antibiotics
11. 
 


SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
12. Add 20g of LB broth Lennox into 1 liter of distilled water in an Erlenmeyer flask
SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
13. Autoclave the LB broth in the Flask on a wet cycle for 30min
SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
14. Storage of LB in 4ºC is recommended and is good for up to 1 year
SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
15. Ensure that the LB broth has come to room temperature before you add ampicillin
SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
16. The final concentration of ampicillin in the broth should be 50μg/mL
SECTION: Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
17. LB broth containing ampicillin should be stored at 4ºC for up to 1 month
SECTION: Preparation of Solid Media
18. LB-Amp Agar and LB-Blasticidin Agar: Each Petri Dish takes about 10mL of LB-Agar so scale the volume accordingly.

1 LB agar tablet makes 50mL
SECTION: Preparation of Solid Media
19. Take 1 tablet and dissolve it in 50mL of distilled water in an autoclaved flask
SECTION: Preparation of Solid Media
20. Autoclave the LB agar for 30min on a wet cycle
SECTION: Preparation of Solid Media
21. Once the cycle is complete check to make sure that all the agar is dissolved
SECTION: Preparation of Solid Media
22. Allow the LB agar to cool until it is comfortable to the touch (50ºC), a water batch set to 50ºC is useful for this step
SECTION: Preparation of Solid Media
23. While the agar is cooling label the plates using sharpie for each antibiotic
SECTION: Preparation of Solid Media
24. The final concentration of ampicillin and Blasticidin should be 50μg/mL
SECTION: Preparation of Solid Media
25. Make sure that the LB agar has cooled to 50ºC as excessive heat will degrade the antibiotics
SECTION: Preparation of Solid Media
23.1. a. Ampicillin = Red 
b. Blasticidin = Blue
TITLE: Molecular Observation Network (MONet)
AUTHORS: maggie.bowman, Alexis Heath, Tamas Varga, Anil Battu, Will Kew, Cheng Shi, Rosey Chu, Che Clendinen, Odeta Qafoku, Jason.Toyoda, qian.zhao, izabel.stohel, Rey Hauchambe, Michael Rosenstock, Albert Lawver, nicholas.sconzo, James Anderson, Patricia Miller, andrew.townsend, Nancy Hess, John Bargar, emily.graham

[DESCRIPTION]
EMSL is leading the effort to develop a national network of environmental sampling and sensing sites to produce comprehensive molecular-level information on the composition and structure of soil, water, and resident microbial communities required to advance the span and accuracy of multiscale models of Earth systems.
Through the Molecular Observation Network (MONet), EMSL will collaborate with a broad range of partners managing an expanding network of selected natural, urban, and managed watershed, coastal, continental, and atmospheric sites, both experimental and observational. 
To establish MONet for the Biological and Environmental Research user community, research will focus on seven areas. Research focused under current development include:
Establishing the supporting field and experimental networks,
Advancing methods for model–data exchange and multiscale modeling,
Automating organic matter and soil analysis,
Developing the next generation of rhizosphere sensors and other field sensors for plants, microbes, nutrients, biomarkers, and aerosols.
The data and models generated through MONet will improve prediction of ecosystem function and response to disturbances, supporting the long-term U.S. Department of Energy goal of scientifically informed decision-making regarding the nation’s energy and environmental security and sustainability. 
For more information please visit: https://www.emsl.pnnl.gov/monet

[STEPS]
TITLE: DNA extraction from mouthwash samples 
AUTHORS: Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Aashish Jha

[DESCRIPTION]
DNA extraction

[BEFORE_START]
Set centrifuge to 4ºC
Keep CD2 solution on ice
Prepare and label collection tubes, microcentrifuge tubes, and MB spin columns

[STEPS]
1. Thaw mouthwash samples on ice for 30 min 

1.1. Transfer desired volume into 1.5 mL or 2 mL eppendorfs
1-2 mL 

2. Centrifuge transferred samples at maximum speed at 4 ºC
2.1. Repeat step 1.1 if more volume is needed to see a pellet or a high yield of DNA is required
3. Discard the supernatant carefully without disturbing the pellet
4. Add 800 µL CD1 and vortex to resuspend pellet

5. Transfer entire eppendorf content to PowerBead Pro tubes
6. Secure PowerBead Pro tubes onto the bead beater and run at maximum speed for 5 min  

7. Centrifuge the PowerBead Pro tubes at maximum speed for 1 min 

8. Transfer the supernatant carefully without disturbing the pellet, into a clean 2 mL microcentrifuge tube
9. Add 200 µL CD2 into the 2 mL microcentrifuge tube and vortex for 10 s 

10. Centrifuge the 2 mL microcentrifuge tube at maximum speed for 1 min 
11. Transfer the supernatant carefully without disturbing the pellet, into a clean 2 mL microcentrifuge tube
12. Add 600 µL CD3 into the 2 mL microcentrifuge tube and vortex for 10 s 
13. Load 650 µL of the lysate onto MB spin columns and centrifuge at maximum speed for 1 min 
13.1. Discard flow-through and repeat step 13 to consume all the lysate
14. Place spin column onto new collection tube, add 500 µL EA, and centrifuge at maximum speed for 1 min 
15. Discard flow-through and place spin column back into the collection tube
16. Add 500 µL CD5 onto spin columns and centrifuge at maximum speed for 1 min 
17. Discard flow-through and place spin column back into the collection tube
18. Centrifuge at maximum speed for 2 min and place spin column into 1.5 mL eppendorf tube
19. Add 50-100 µL nuclease-free water to the center of the spin column and leave at room temperature for around 7 min 
20. Centrifuge at maximum speed for 1 min, measure with Qubit and flash freeze DNA with liquid N2
TITLE: Intraganglionic injection of AAV into nodose ganglia in mice
AUTHORS: Seol-Hee Kim, patil, Thomas Taylor-Clark

[DESCRIPTION]
We have developed a novel method of injecting AAV vectors directly into the sensory ganglia, vagal and dorsal root ganglia, of mice. This technique provides for the first time a tool for expression of specific markers in sensory neurons that aid in the tracing of peripheral sensory innervation of organs like the airways and the esophagus.

[STEPS]
2. Approximately 2 cm of incision was made over a shaved superficial portion of the masseter muscle area. 
1. Mice were anesthetized with a mixture of ketamine (50 mg/kg) and dexmedetomide (0.5 mg/kg) via intraperitoneal injection.
4. The vagal nodose ganglia was carefully exposed. 
5. Micropipettes were filled with AAV (0.7ul volume) using capillary force. The virus microinjection assembly consisted of a pulled glass micropipette (~10-15μm tip diameter) attached to a 1 mL syringe via plastic tubing. 
3. The skin was retracted, and the salivary glands separated gently without cutting them off of the mouse entirely. The vagus nerve, which runs next to the common carotid artery, was identified. The vagus nerve was separated from the common carotid artery and the anterior laryngeal nerve using a cotton tip. 
6. The tip of the micropipette was gently inserted into the nodose ganglia and then injected by depressing the plunger (∼0.5 p.s.i.). 
7. After AAV injection, the mice received atipamezole (5 mg/kg) via subcutaneous injection for rapid recovery. The mice were injected with meloxicam (500 mg/kg via subcutaneous injection) as a post-analgesic on the day and 24 hrs later.
8. 4 weeks later, mice were euthanized by CO2 asphyxiation for tissue collection.
TITLE: Silver Stain Gel for IP-TDMS Development
AUTHORS: Lauren  Adams

[DESCRIPTION]
Silver stain gels can be used to examine total protein in a given sample. This method is less sensitive than western blotting, but typically more sensitive than Coomassie blue staining. 

[STEPS]
1. Take aliquots from IP fractions and add sample loading buffer to a final concentration of 1X. Boil samples in hot plate at 95ºC for 5-10 min. Briefly, centrifuge samples to ensure sample entire sample is towards bottom of the tube.
2. Open gel and place into gel box. Add 1 x MES running buffer, ensuring that there are no bubbles within the wells of the gel. Carefully load samples, without loss of sample from the well. Make sure that the gel wells have a large enough capacity to contain the entire sample volume. Load at least one lane with 2-3 μL of protein MW ladder. Run the gel at 120-150 V for approximately 40 minutes, or until the sample dye has reached the bottom of the gel. 
3. Remove the gel from the cassette and gently wash with deionized water. Cut off the bottom of the gel (where there is a ridge). Place get into clean gel box. Wash gel in MilliQ (MQ) H2O 3 times for 5 minutes each.
4. Cover with Fixing Buffer for ≥ 20 minutes.
5. Wash in wash buffer for 10 minutes.
6. Wash in MQH2O for 10 min. 
7. Treat with sensitizing buffer for 1 minute. 
8. Wash twice in MQH2O for 1 minute each.
9. Stain AgNO3 for 20 minutes; after this step, all waste must be collected in hazardous waste container. 
10. Wash twice in MQH2O for 20 seconds each.
11. Expose for 1 minute in developing buffer; watch the gel avoid overdeveloping. 
12. Treat with terminating buffer for 1 minute; make sure the gel is no longer being developed.
13. Image the gel.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>PNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. The enzyme will remain fully active under reaction conditions (37˚C) for at least 96 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.</p>

[STEPS]
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TITLE: HTTM : DNA Extraction
AUTHORS: Antoine Champie, Amélie De Grandmaison

[DESCRIPTION]
Part two of the HTTM protocol. A low-cost and high-throughput Tn-seq protocol. 
This part cover the DNA extraction from cell pellets of transposon insertion mutants and subsequent silica columns regeneration. 

[STEPS]
SECTION: DNA extraction
1. Prepare the lysis solution by adding 165 µL of proteinase K to 66 mL of homemade lysis buffer and mix well. 

SECTION: DNA extraction
2. Add 600 µLof lysis solution to each well of the deep-well plate and resuspend the pellet. 

SECTION: DNA extraction
3. Cover with an adhesive aluminum cover and incubate at 55 °C for 60 min. 

SECTION: DNA extraction
4. While still warm, add 260 µLof ethanol 100%, without overmixing.

 

SECTION: DNA extraction
5. Transfer immediately to a deep-well plate fitted with an array of silica columns. 
SECTION: DNA extraction
6. Centrifuge twice at 4000 x g, 10 min . 


SECTION: DNA extraction
7. Discard flowthrough and add 500 µL of wash solution.

SECTION: DNA extraction
8. Centrifuge at 3000 x g, 10 min .


SECTION: DNA extraction
8.1. Repeat steps 7 and 8. 
SECTION: DNA extraction
9. Discard flowthrough.

SECTION: DNA extraction
10. Centrifuge at 3000 x g, 10 min  to eliminate traces of wash solution. 
SECTION: DNA extraction
11. Discard flowthrough.

SECTION: DNA extraction
12. Add a collector plate between the silica column array and the deep-well plate. 
SECTION: DNA extraction
13. Add 50 µLof low TE to the silica matrix in each well. 

SECTION: DNA extraction
14. Cover with an adhesive aluminum foil and incubate at55 °C for 15 min.

SECTION: DNA extraction
15. Centrifuge at 3000 x g, 10 min .

SECTION: Silica array regeneration (Optional)
16. Put the contaminated silica array on an empty deep-well plate.
Add 150 µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well. 

SECTION: Silica array regeneration (Optional)
17. Incubate at Room temperature for 5 min.

SECTION: Silica array regeneration (Optional)
18. Centrifuge 3000 x g, 5 min .

SECTION: Silica array regeneration (Optional)
19. Add 200 µL of 1.5N HCl+ 0.15% (v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
20. Incubate at Room temperature for 30 min .

SECTION: Silica array regeneration (Optional)
21. Centrifuge 3000 x g, 5 min .

SECTION: Silica array regeneration (Optional)
22. Add 150 µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
23. Incubate at Room temperature for 5 min.

SECTION: Silica array regeneration (Optional)
24. Centrifuge 3000 x g, 5 min.
SECTION: Silica array regeneration (Optional)
24.1. Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through. 
SECTION: Silica array regeneration (Optional)
25. Add 200 µLof ddH2O to each well. 

SECTION: Silica array regeneration (Optional)
26. Centrifuge 3000 x g, 5 min.
SECTION: Silica array regeneration (Optional)
26.1. Repeat steps 25 and 26.
SECTION: Silica array regeneration (Optional)
27. Silica columns array are ready to be reused.
TITLE: Bead-based normalization for NGS
AUTHORS: Tomasz Suchan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bead-based normalization protocol for genomic libraries based on Hosomichi et al. (2014). It uses diluted AMPure or other SPRI beads solution in order to bind a small amount of DNA from each sample.</div></div>

[STEPS]
?. [Preparation]
Prepare a solution of 20% PEG, 2.5 M NaCl in TE buffer by mixing:5 ml of 5 M NaCl0.83 ml of molecular-grade water100 ul of 1 M Tris20 ul of EDTA50 uL of 10% Tween 104 ml of 50% PEG-8000
?. [Preparation]
Dilute AMPure or homemade SeraMag beads 20-fold in the above solution (for instance, add 10 ul of AMPure to 190 ul of the solution).
?. [Normalization]
Add 20 ul of DNA library to the tube.
?. [Preparation]
Prepare 2.5 M solution of NaCl:place a 50 ml Falcon tube on the scale and tareadd 14.61 g of NaCl to the tubeadd water to 40 mldissolve NaClfill up the tube with water to 50 ml and mix
?. [Preparation]
Prepare 50% solution of PEG-8000:place a 50 ml Falcon tube on the scale and tareadd 12.5 g of PEG-8000 to the tubeadd 14 ml of nuclease-free waterclose the tube, and dissolve PEG by mixing vigorously, preferably on an orbital shaker with heatingfill up the tube with water to 25 ml and mix gently by flipping the tube to avoid bubbles
?. [Normalization]
Add 20 ul of the diluted beads.
?. [Normalization]
Add 20 ul of isopropanol.
?. [Normalization]
Mix well by pipetting.
?. [Normalization]
Incubate for 5 minutes at room temperature.
[DNA binding]
?. [Normalization]
Place the tube on the magnetic stand and wait until the beads separate.
?. [Normalization]
With the tube still on the magnet, remove and discard the supernatant.
?. [Normalization]
Add 10 ul of 80% ethanol and incubate for 30 sec. at room temperature.
[EtOH wash]
?. [Normalization]
Carefully remove all the ethanol and discard it.
?. [Normalization]
Repeat steps 13 and 14.
?. [Normalization]
Leave the tube on the magnetic rack and let the beads dry. Do not overdry the beads (the pellet should not be shiny anymore but there should not be cracks on it).
?. [Normalization]
Add 20 ul of molecular-grade water on the beads. Make sure that the beads are wet before removing the tube from the magnet!
?. [Normalization]
Remove from the magnet and mix the beads with water by pipetting.
?. [Normalization]
Incubate 10 minutes at room temperature.
[DNA elution]
TITLE: Intracerebroventricular administration of compound in Mus Musculus
AUTHORS: Jean-Louis Parmasad

[DESCRIPTION]
Intracerebroventricular administration of compound in Mus Musculus. Here we utilize the newly developed CDK14 covalent inhibitor, and inject it into lateral ventricle of Mus Musculus brain.

[STEPS]
SECTION: Day before surgery
1. 16h before stereotactic surgeries, load Alzet‱ Mini-Osmotic Pumps (model 2004) with 1.47μg/uL of FMF-04-159-2 (R&D Systems 7158, Minneapolis, MN, USA in vehicle solution containing 8% DMSO (Fisher Scientific, BP231, Hampton, NH, USA), 2% Tween 80 (Fisher Scientific, BP338-500) and 90% ddH2O). These pumps administer the solution at 0.35mg/kg/day.
SECTION: Day before surgery
2. Trim brain infusion catheters to 3.5cm from Alzet‱ Brain Infusion Kit and attach to pump as per manufacturer’s instructions.
SECTION: Day of surgery
4. Deeply anesthetize mouse with isoflurane.
SECTION: Day before surgery
3. Incubate brain infusion assemblies at 37 °C in sterile saline until implantation. 
SECTION: Day of surgery
5. Implant canula at the appropriate location (coordinates relative to bregma: −1.1 mm medial-lateral; −0.5 mm antero-posterior and −3 mm dorso-ventral). 
SECTION: Day of surgery
6. Secure cannula using dental cement.
SECTION: Day of surgery
7. Bury the pump in a subcutaneous pocket of the mouse’s back. 
SECTION: Day of surgery
8. Allow for 28 days with the brain infusion catheter implanted.
SECTION: Day of surgery
9. Monitor mouse body weight, activity, neurological signs, facial grimace, coat condition, and respiration within 28 days after the surgery.
SECTION: Day of surgery
10. Sacrificed mice, collect organs on the 28th day of the administration period.
TITLE: Immunofluorescence staining for postmortem mouse brain tissue
AUTHORS: Elizabeth P.

[DESCRIPTION]
This is a basic protocol for staining mouse brain tissues using immunofluorescence and immunohistochemistry techniques. 

[STEPS]
SECTION: Preparation
1. For this protocol I recommend staining slices in a 12-well plate, and washing the slices in a separate 24-well plate. Each well can hold one solution or wash for the slices (about 1mL of solution in each well should be fine). Be sure to label the plate with the contents of each well.
SECTION: Staining
4. Wash the tissue slices in 1x PBS for ten minutes at room temperature for 3 total washes.
SECTION: Staining
5. Place the tissue slices in blocking solution for 30 minutes on the shaker at room temperature.
SECTION: Staining
6. Move the tissue slices to the primary antibody solution and leave to shake gently overnight at 4 degrees celsius.
SECTION: Staining
7. Wash the slices in 1x PBS plus 0.05% Tween, 4 times for 5 minutes at room temperature.
SECTION: Staining
8. Place the tissue slices in the fluorescent secondary antibody solution, shake gently for 2 hours at room temperature, making sure to cover the slices and their solution to avoid bleaching the fluorescence.
SECTION: Staining
9. Wash the slices in 1x PBS plus 0.05% Tween, 4 times for 5 minutes at room temperature.
SECTION: Staining
10. Wash the slices one final time in 1x PBS for 5 minutes at room temperature.
SECTION: Staining
11. Mount the tissues immediately.
SECTION: Preparation
2. Prepare the blocking solution ahead of time: 5% Normal Donkey Serum in PBS1x with 0.3% TritonX. For 10mL of solution, add 9.5 mL of the PBS + TritonX solution and 0.5mL of NDS.
SECTION: Preparation
3. The antibody solutions can also be prepared ahead of time, they are created by simply adding the antibodies to aliquots of the blocking solution prepared above. The concentration of the primary and secondary antibody solutions will vary depending on the specific antibody and desired target of the staining; a solution of 1:250 or 4uL/mL concentration is recommended for first-time antibody stains.