eamag/protocols-with-steps huggingface dataset:
TITLE: AxyPrep magnetic Bead normalization and PCR clean up For dual index
AUTHORS: Alexander  J Bradshaw

[DESCRIPTION]
In-house protocol used to prepare PCR amplicons with AxyPrep beads for Illumina sequencing.

[STEPS]
1. Place Elution buffer, Magnetic Beads, and binding buffer on the bench and allow each to reach room temperature before using.

Room temperature 

2. Mix magnetic beads until homogenous (no seriously, vortex the shit out of them)
3. Add 10ul of magnetic beads to each well. Pipette Up and down 5 times to mix completely


(10ul collects 200ng of DNA, adjust accordingly if more or less is desired)  
4. add 100 ul of binding buffer (BB) to each well. Pipette Up and down 5 times to mix completely
5. Incubate samples at room temperature for 10 minutes

25 Room temperature 10 min 

6. place on magnetic separation device for 4 minutes or until the solution clears

4 min 

7. with the sample plate still, on the magnet, remove and discard the supernatant by pipetting.
8. Remove the sample plate from the magnetic separation device. 

Add 150 μl freshly prepared 70% ethanol to each well 

pipet mix 5 times and incubate for 2 minutes at room temperature.

2 min 

9. Place the sample plate on the 96 magnetic separation device for 4 minutes or until the solution clears.

 4 min 

10. With the sample plate still on the magnet, remove and discard the supernatant by pipetting.
11. Dry the beads by incubating the plate for 2 minutes at room temperature with the plate still on the magnetic separation device.

.Remove the sample plate from the magnetic separation device. 

2 min Room temperature 

12. Add 50μl of EB-N Elution Buffer to each well and pipet up and down 5 times to mix.

Incubate the sample plate for 2 minutes at room temperature.
2 min Room temperature 

13.  Place the sample plate back on the magnetic separation device and wait 4 minutes or until the magnetic beads clear from the solution.  

4 min 


14. Transfer the eluate (cleared supernatant) to a new plate/tube for storage or for subsequent applications.

For us, we will pool all 50ul of each sample into a single tube. If you have a lot of samples this could be a 50mL conical tube
TITLE: Enteric neuron activity during spontaneous motor complexes in mouse colon
AUTHORS: Kristen Smith-Edwards

[DESCRIPTION]
This protocol describes the steps to prepare mouse colon and image GCaMP calcium activity in myenteric neurons during spontaneous colonic motor complexes. 

[STEPS]
1. Euthanize EIIa-GCaMP mice (offspring from the pairing of B6.FVB-Tg(EIIa-cre)C5379Lmgd/J [RRID:IMSR_JAX:003724;cat. 003724; Jax Labs] and B6J.Cg-Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/MwarJ [RRID:IMSR_JAX:028866;cat. 028865; Jax Labs]), by inhalation of isoflurane and thoracotomy. Remove the entire colon from mouse and place into a Sylgard-lined Petri dish (Dow Corning). Circulate oxygenated artificial cerebral spinal fluid at room-temp (ACSF, containing in mM: 117.9 NaCl [cat. S9888, Sigma], 4.7 KCL [cat. P3911; Sigma], 25 NaHCO3[catS6014; Sigma], 1.3 NaH2PO4, [cat. S8282; Sigma] 1.2 MgSO47H2O [cat. 230391; Sigma], 2.5 CaCl2 [cat. C1016; Sigma], 11.1 D-glucose [cat. G5767; Sigma], 2 sodium butyrate [cat. B5887; Sigma], and 20 sodium acetate [cat. S2889; Sigma]). Cut the colon open longitudinally and pin flat using minutien pins (Fine Science Tools; cat. 26002-20) with mucosal side facing down. A small amount of stretch should be applied when pinning the colon for optimal imaging conditions. The full-length, full-thickness colon should remain intact for imaging activity during spontaneous motor complexes. Transfer to the stage of an upright fluorescence microscope (DM6 FS, Leica) equipped with camera (Prime 95B, Photometrics, Roper Scientific) and software (Metamorph, Molecular Devices) for calcium imaging and slowly heat up circulating fluid to 35-37 C using a heated water bath.
2. After the colon has warmed up and equilibrated (10-15 minutes), use a 10X objective, focus on the myenteric neurons and image calcium signals during spontaneous motility behavior. Myenteric neurons can be distinguished from other cell types based on 1) their location in ganglia within the myenteric plexus between the longitudinal and circular muscle layers, 2) cell body shape and size, and 3) the kinetics of their calcium transients. Set imaging parameters to 150ms exposure time, 1200 frames, 2x2 binning to obtain 3-min duration imaging files. Collect several consecutive 3-min imaging files for a total of 9-15 min to capture multiple colon motor complexes (CMCs), which typically occur every 2-5 min depending on the amount of stretch applied to the colon tissue. 
3. Export imaging files to ImageJ and open as a multi-TIFF stack. Use the ImageJ plugin "Template Matching, Align Slices in Stack" to quantify and correct for tissue displacement in the x and y axis of the field of view. Save the x and y displacement values for all 1200 frames, convert frames to time by multiplying the frame by 0.150, and plot as a motility trace (displacement over time) in Excel. 
4. After imaging files have been corrected for movement in ImageJ and displacement data saved, myenteric neuron activity can be analyzed by drawing regions of interests over cell bodies and plotting the mean intensity changes over time.
TITLE: PMMA Spin-coating
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to spin coat a thin layer of PMMA on a glass cover slip.</div></div>

[STEPS]
?. [Prepare surface]
Wipe cover slip with 70% ethanol using a delicate task wipe.
?. [Spin-coat]
Place cove slip into spin coater, centering with the square in the spin coater.
?. [Spin-coat]
Dispense PMMA solution onto the center of the cover slip.
30 µl
?. [Spin-coat]
Spin for at 2000 rpm.
This step can be varied, where higher angular velocities will form thinner coatings.
?. [Bake substrate]
Carefully remove cover slip from spin coater using tweezers.
?. [Bake substrate]
Place cover slip onto hot plate for .
TITLE: Intra-cardiac mouse perfusion
AUTHORS: Lauren C. Faget, Thomas Hnasko

[DESCRIPTION]
Hnasko lab intra-cardiac mouse perfusion protocol

[STEPS]
SECTION: Prepare subject
1. Anesthetize with a lethal dose of pentobarbital (200 mg/kg i.p. or 0.1-0.2 ml at 10 mL/kg in 0.9% NaCl).
SECTION: Prepare subject
2. After 2-3min, verify that the animal is completely anesthetized by checking for absence of response to hind paws / tip of tail pinch using nails or small tweezers.
SECTION: Prepare subject
3. Once fully anesthetized, affix the animal to a silicone dissection pad (dissection pins through paws) in a splay position with ventral side up. Place silicone pad on a grid panel over a container for blood and PFA collection. Tilt the grid/pad slightly so that blood/PFA flows backward off the pad into the container. Work inside a chemical fume hood to prevent PFA inhalation.
SECTION: Procedure
4. Cut skin transversally at the base of the thorax.
SECTION: Procedure
5. Then cut muscle transversally at the same level.
SECTION: Procedure
6. Slightly lift the thorax by pulling on the xiphoid process (white tip of the thoracic body). Do not lift too much to not damage inner organs.
SECTION: Procedure
7. Cut the diaphragm with fine scissors (Be careful to not puncture the heart or lungs at this step).
SECTION: Procedure
8. Cut the thorax laterally on both sides (Be careful to not puncture the heart or lungs at this step too), pull backward, and fix thorax to pad with a needle on the left side of the animal.
SECTION: Procedure
9. Clear heart from connective tissues.
SECTION: Procedure
10. Place butterfly needle (connected to perfusion pump – prefilled with ice cold 1X PBS) in the tip of the left ventricle (darker and longer side of the heart – prominent tip). Be careful to not puncture through the right ventricle or the left atrium by going too deep. Cut the right atrium (darker atrium) to allow blood/perfusate to escape circulation.
SECTION: Procedure
11. Clamp the butterfly needle in place using a hemostat (or hold in place and clamp in next step).
SECTION: Procedure
12. Start the pump using a flow rate of 5-6 mL/min. Perfuse with ~10 mL of ice-cold 1X PBS (~2 min).


SECTION: Procedure
13. Blood should come out of the right atrium with a regular flow (If you see clear liquid right away, you might have punctured through the heart – can try to slightly pull back the needle, but you may have a failed perfusion). Clamp the butterfly needle in place if you did not in previous step.
SECTION: Procedure
14. When blood has been depleted (~2min) switch to ice-cold freshly made 4% paraformaldehyde (PFA) pH 7.4 for 5 min to 8 min(30-50 mL).
    
SECTION: Procedure
15. Remove brain or other tissue of interest.
SECTION: Procedure
16. Post-fix tissue submerged in 4% ice-cold PFA for 120 min,  , or as appropriate for intended use.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes the preparation of and treatment with DNase I to degrade DNA in solutions containing iron chloride, EDTA&ndash;Mg ascorbate buffer, and cesium chloride. DNase I treatment, CsCl purification, and sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012.&nbsp; We use this protocol primarily to remove free host and other contaminating DNA from phage preparations prior to extracting nucleic acid from the phage particles.

[GUIDELINES]
<strong>Resuspending DNase I from powder</strong><br />Determine number of units per mg powder: Roche grade II (cat.no. 10 104 159 001) contains approximately 2,000 Kunitz units per mg and comes in 100 mg size which equals 200,000 U. Resuspend in 5ml NEB recommended storage buffer, 10 mM Tris-Cl pH 7.5, 2 mM CaCl2 in 50% glycerol which equals 40,000 U/ml and store at -20&deg;C .<br /><br /><strong>Unit definition</strong><br />One unit is the amount of enzyme which will completely degrade 1&mu;g of pBR322 DNA in 10 min at 37&deg;C in DNase I reaction buffer.<br /><br /><strong>Reaction Buffer</strong><br />Prepare a 10x reaction buffer containing MgCl2 and CaCl2 which are required for enzymatic activity. Final 1x buffer contains 10 mM Tris-HCl pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2.<br /><br /><strong>DNase I reaction conditions</strong><br />DNase I was tested for ability to degrade DNA in solutions containing iron chloride, EDTA-Mg ascorbate buffer and cesium chloride (see BTP Exp. 72) under conditions of 37&deg;C for 1 hr, room temperature for 2 hr and 4&deg;C overnight. The DNA concentration (1 Kbp DNA ladder) in each solution was 200 ng/&mu;l. The DNase I (40,000 U/ml) was diluted 1:40 in 10x reaction buffer with final concentration of 1,000U/ml (=1 U/&mu;l) in 10x buffer. The diluted DNase I was used at a 1:10 dilution in the DNA solutions: 1 &mu;l DNase I in 9 &mu;l DNA. The DNA was degraded in each solution at all the reaction conditions tested.<br /><br /><strong>DNase I Storage Buffer: 10 mM Tris-HCl, pH 7.5, 2 mM CaCl2 in 50% glycero</strong>l<br />100 &mu;l 1 M Tris-HCl, pH 7.5 (autoclaved or filter-sterilized stock)<br /> 20 &mu;l 1 M CaCl2 (filter-sterilized stock)<br /> 10 ml 50% Glycerol (autoclaved)<br /><br /><strong>10X DNase I Reaction Buffer: 100 mM Tris-HCl, pH 7.6, 25 mM MgCl2, 5 mM CaCl2</strong><br />1 ml 1 M Tris-HCl, pH 7.5 (autoclaved or filter-sterilized stock)<br />250 &mu;l 1 M MgCl2 (filter-sterilized stock)<br /> 50 &mu;l 1 M CaCl2 (filter-sterilized stock)<br />8.7 ml Q-water (autoclaved)<br /><br /><strong>Citation:&nbsp;</strong>​Hurwitz, B. L., Deng, L., Poulos, B. T., &amp; Sullivan, M. B. (2012). Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics. Environmental Microbiology. doi:10.1111/j.1462-2920.2012.02836.x

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: DNA extraction from fecal samples
AUTHORS: Yoshiyuki Matsuo

[DESCRIPTION]
DNA extraction method for metagenomic sequencing of the gut microbiota

[STEPS]
SECTION: Preparation of fecal samples
1. Place 50-100 mg of fecal sample into tube.
SECTION: Preparation of fecal samples
2. Add 1 mL of PBS per 100 mg of feces.

SECTION: Preparation of fecal samples
3. Mix thoroughly by vortexing.
SECTION: Preparation of fecal samples
4. Allow the sample to stand for 2 min to sediment large debris.

SECTION: Preparation of fecal samples
5. Transfer 300 µL of the suspension to 1.5 mL tube.

SECTION: Preparation of fecal samples
6. Centrifuge at 10000 x g, 3 min.

SECTION: Preparation of fecal samples
7. Discard the supernatant.
SECTION: Preparation of fecal samples
8. Resuspend the pellet (~30 mg of feces) in 300 µL of PBS.

SECTION: Preparation of fecal samples
9. Incubate at 70 °C for 10 min on the block heater.

SECTION: Mechanical cell disruption by bead beating
11. Transfer 300 µL of the suspension to EZ-beads tube.

 

SECTION: Mechanical cell disruption by bead beating
12. Lyse cells either by using disruption device (12.1) or vortex mixer (12.2).
SECTION: Mechanical cell disruption by bead beating
12.1. Place the EZ-beads tube in Micro Smash instrument and disrupt cells by shaking at  . 
 

 


SECTION: Mechanical cell disruption by bead beating
12.2. Place the EZ-beads tube on MN Bead Tube Holder attached to Vortex-Genie mixer and vortex for 5 min at maximum speed.
 

SECTION: Mechanical cell disruption by bead beating
13. Briefly spin the tube to collect contents.
SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
14. Add 300 µL of Lysis Buffer and 30 µL of Proteinase K Solution to the sample in EZ-beads tube.

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
15. Mix by inverting the tube.
SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
17. Incubate at 56 °C for 20 min on the block heater.

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
18. Briefly spin the tube.
SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
19. Transfer the supernatant (~500 µL ) to 1.5 mL tube.

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
20. Centrifuge at 18000 x g, 3 min.

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
21. Transfer the cleared lysate to Maxwell RSC Cartridge.
SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
22. Add 50 µL of Elution Buffer to elution tube.

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
23. Start the extraction run following the manufacturer's instructions.
 

SECTION: Preparation of fecal samples
10. Cool to Room temperature. 

SECTION: Automated DNA extraction using Maxwell RSC Blood DNA Kit
16. Briefly spin the tube.
TITLE: Coil construction
AUTHORS: Clarice D. Aiello

[DESCRIPTION]
To systematically quantify magnetic field effects in cell biology, it is crucial to produce fields that can be automatically adjusted and that are stable throughout an experiment's duration, usually operating inside an incubator. Here, we report on the design of a fully automated 1D Helmholtz coil setup that is internally water cooled, thus eliminating any confounding effects caused by temperature fluctuations. The coils also allow cells to be exposed to magnetic fields from multiple directions through automated controlled rotation.

This protocol accompanies arXiv manuscript http://arxiv.org/abs/2406.19555.

[STEPS]
SECTION: Materials
1. Here are the materials required and the assembly instructions for the coils.

* Copper wire (0.71 mm diameter);
* 3D-printed coil holder in thermoplastic material (CAD files found here);
* Machined coil enclosure made out of Delrin or aluminum (CAD files also found here); 
* Thermally conductive glue (e.g., 10 g of GENNEL G109);
* Two-part epoxy (e.g., J-B Weld 8276);
* Silicone sealant (e.g. , Aqueon);
* Multi-axes magnetometer for testing (e.g., F71 Multi-axis sensor).


SECTION: Assembly instructions
2. 
 

* 3D-print the holder around which the coils are wound, and machine the coil enclosure. Choose a material (e.g., a thermoplastic) that is sturdy, lightweight, and corrosion-free;
* A. Wind the copper wire 96 times around the 3D-printed holder. Secure the coils' surfaces using the thermally conductive glue;
* B. Place the wound coils and their holder inside the coil enclosure and establish the necessary electrical connections. To seal the electrical connections, apply the two-part epoxy;
* C. Prevent water leakage by applying the silicone sealant before screwing the caps of the coil enclosure. Firmly fix the caps to the coil enclosure to ensure a watertight seal;
* D. Use the multi-axes magnetometer to measure the produced magnetic fields when different currents are applied.
TITLE: Swedish Traditional Gingersnaps
AUTHORS: Angelina Spotts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h2><span style = "font-weight:bold;">Ingredients</span></h2></div><div class = "text-block"><h2></h2></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">3/4 cup butter, softened</li><li style = "counter-reset:ol0;list-style-type:disc;">1 cup sugar</li><li style = "counter-reset:ol0;list-style-type:disc;">1 large egg, room temperature</li><li style = "counter-reset:ol0;list-style-type:disc;">1/4 cup molasses</li><li style = "counter-reset:ol0;list-style-type:disc;">2 cups all-purpose flour</li><li style = "counter-reset:ol0;list-style-type:disc;">2 teaspoons baking soda</li><li style = "counter-reset:ol0;list-style-type:disc;">1 teaspoon ground cinnamon</li><li style = "counter-reset:ol0;list-style-type:disc;">1 teaspoon ground cloves</li><li style = "counter-reset:ol0;list-style-type:disc;">1 teaspoon ground ginger</li><li style = "counter-reset:ol0;list-style-type:disc;">1/4 teaspoon salt</li><li style = "counter-reset:ol0;list-style-type:disc;">Additional sugar</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Directions</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">In a bowl, cream butter and sugar. Beat in egg and molasses. Combine the flour, baking soda, cinnamon, cloves, ginger and salt; gradually add to creamed mixture. Chill.</li></ul></div><div class = "text-block"><h2><span style = "font-weight:bold;">Directions</span></h2></div><div class = "text-block"><h2></h2></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block">Roll into 1-1/4-in. balls and dip into sugar. Place 2 in. apart on ungreasedbaking sheets. Bake at 375° for about 10 minutes or until set and surface cracks. Cool on wire racks.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), a <a href="https://www.creative-biolabs.com/car-t/cellrapeutics-chimeric-antigen-receptor-car-technology.htm" target="_blank" rel="noopener noreferrer">chimeric antigen receptor T cell</a> immunotherapy, has been in use for many years but has only been used in clinical practice in recent years. It has significant curative effect in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered as one of the most promising approaches to treatment of cancer. As with all technologies, CAR-T technology has also undergone a long evolutionary process. It is during this series of evolution that CAR-T technology has gradually matured.</p>
<p> </p>

[STEPS]
?.
TITLE: Gentamicin Protection assay (Intracellular Survival Assay) for Salmonella Typhimurium/Typhi
AUTHORS: Kasturi  Chandra

[DESCRIPTION]
Salmonella invades the epithelial cells by inducing bacterial uptake, whereas phagocytes readily take up the bacteria by phagocytosis. Intracellular Salmonella is resistant to gentamicin, but extracellular bacteria are susceptible to the antibiotic, therefore, adhesion, invasion, and bacterial replication can be measured using this protocol. Inside the cells, Salmonella proliferates and the rate of proliferation can be determined by enumerating the bacterial CFU from the infected cells.

[STEPS]
1. Epithelial cells were infected with mid-log phase culture of bacteria grown in LB (OD600 0.3)

OR

phagocytes were infected with overnight culture (OD600 0.3). The multiplicity of infection (MOI) of 10 was used in each case.
2. Bacterial attachment to host cells was enhanced by centrifuging at 600 rpm for 10 min.
3. After 25 min of infection, cells were washed three times with 1x PBS and treated with gentamicin (100 μg/ml in complete media) for 1 hour and then maintained with 25 μg/ml gentamicin for the rest of the experiment.
4. 0.1% Triton-X 100 (v/v in 1x PBS) was used to lyse the cells and the lysate was plated on Salmonella-Shigella (SS) agar for S. Typhimurium strains and Wilson Blair (WB) agar for S. Typhi strains.
6. For invasion assay:
The cells were washed three times with 1x PBS and lysed after incubation in 100 μg/ml gentamicin treatment (i.e. 1 hour post infection) and percent invasion was calculated with respect to the pre-inoculum used for infection.
7. For intracellular survival assay (ICSA):
The cells were washed three times with 1x PBS and lysed at 2 hours and 18 hours post infection. CFU at 18 hours was divided by CFU at 2 hours to obtain fold replication of the intracellular bacteria.
5. For adhesion assay:
The cells were washed three times with 1x PBS and lysed immediately after the initial 25 min incubation. The percent adhesion was calculated with respect to the pre-inoculum used for infection.