eamag/protocols-with-steps huggingface dataset:
TITLE: Media changes and Passaging in 2- or 5-layer CellStacks
AUTHORS: Allan JW Lui

[DESCRIPTION]
Protocol for handling CellStacks

[BEFORE_START]
Ensure shelves of incubator are truly level with a spirit level or app

[STEPS]
SECTION: Passaging
6. Per flask, Prepare and pre-warm:
 



SECTION: Passaging
7. Pour out media in flask, wash out remaining media twice by:
Adding PBS
Distributing evenly between layers to cover the entire culture surface area
Pouring out PBS
SECTION: Passaging
8. Add Trypsin
Incubate flask for around 3 min  
Tap flask to dislodge cells
SECTION: Passaging
9. Add 1 aliquot of media for trypsin inactivation and distribute evenly between layers.
Collect cell suspension into duran bottles (250ml for 2-layer, 500ml for 5-layer)
Wash out remaining cells with the other aliquot of inactivation media similarly.

Reduce clumping by pipetting up and down multiple times with a 10ml serological pipette against the bottom of the bottle  
SECTION: Passaging
11. If necessary, centrifuge cells for replating at 200 x g, 5 min and remove supernatant.

If plating into CellStacks: 
Resuspend cells in media for plating
Transfer suspension into flask by pouring or pipetting

SECTION: Passaging
10. Count cells using a Vi-Cell and calculate volume of cells for seeding
SECTION: Media Changes
1. Per flask, Prepare and pre-warm:
 


SECTION: Media Changes
2. Take flask out of incubator, and place in upright position. Wipe down cap areas with 70% ethanol if desired.

Holding the flask with the long side facing downwards, uncap the bottom cap, and pour out media in flask onto a walls of a large 10L tub to prevent splashing, shake flask to remove the final few drops.
  

SECTION: Media Changes
3. Place CellStack on its flat position.

For 2-layer CellStacks, pipette in new warm media 50-60ml at a time until 220ml has been added
For 5-layer CellStacks, pour in 550ml of new warm media (often an entire bottle)
SECTION: Media Changes
4. To evenly distribute media between layers: Flip flask towards you such that it rests on its longer side.
  Then tilt into the upright position:
 
SECTION: Passaging
12. Reseed cells at desired density.

If plating into CellStacks: 
Calculate the required volume of cell suspension for plating
Remove the same volume of media from the pre-warmed aliquot of media for plating and replace with the cell suspension
Transfer suspension into flask by pouring or pipetting


SECTION: Media Changes
5. Return flask to incubator, lying flasks flat while minimising sudden or large movements to avoid media spilling between layers
SECTION: Pelleting cells
13. Transfer desired number of cells (usually 5e7) into 50ml centrifuge tubes
SECTION: Pelleting cells
14. Centrifuge cells at 200 x g, 5 min and remove supernatant. 
Resuspend with 5 mL , centrifuge and remove supernatant again.

SECTION: Pelleting cells
15. Flick tube to loosen pellet and freeze on dry ice.
TITLE: Protocol for T7 Exonuclease (NEB #M0263)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">T7 Exonuclease efficiently degrades nicked and linear dsDNA (with blunt or 3' overhangs) from 5' to 3' direction, leaving supercoiled dsDNA inctact.*</div><div class = "text-block"><span style = "font-weight:bold;">*Note: </span><span>For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.</span></div></div>

[STEPS]
?. Set-up the reaction as follows:  AB1Components50&nbsp;μl REACTION2&nbsp;DNAup to 1&nbsp;μg3&nbsp;NEBuffer 4 (10x)&nbsp;5 μl (1X)4&nbsp;T7 Exonuclease&nbsp;1 μl (10 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
AB1Components50&nbsp;μl REACTION2&nbsp;DNAup to 1&nbsp;μg3&nbsp;NEBuffer 4 (10x)&nbsp;5 μl (1X)4&nbsp;T7 Exonuclease&nbsp;1 μl (10 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
?. Incubate at  for .
25 °C
?. Stop reaction by adding EDTA to at least .
?. To clean up treated samples, we recommend using one of the following steps:a. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030) orb. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), orc. Performing a phenol/chloroform extraction followed by ethanol precipitation.
TITLE: Dinoflagellate transformation
AUTHORS: Lu Wang, Brittany Sprecher, Huan Zhang, Senjie Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Protocol of dinoflagellate cell transformation</span></div></div>

[STEPS]
?. Cultivate Fugacium kawagutii (formerly Symbiodinium kawagutii) cells in L1 medium with an antibiotic cocktail for 3-4 weeks and Alexandrium catenella cells in L1 with antibiotics for 1-2 weeks. (Final antibiotic concentration is 0.1 mg/ml for Ampicillin, 0.05 mg/ml for Kanamycin and 0.05 mg/ml for Streptomycin).
?. Harvest cells by centrifugation at 800g for 5 min at 4°C.
?. For F. kawagutii cells, use 0.1M EDTA to resuspend the cell pellet, centrifuge at 800 g for 2 min at 4°C.
?. Wash cells with 10% Glycerol 3-4 times, centrifuge at 800 g for 2 min in 4°C.
We also use 384mM D-sorbitol and it works well too.
?. Resuspend the pellet in 10% Glycerol with final cell concentration at 106 to 108/ml.
?. Incubate ~40µl of cells with 5µl (~1µg) of DNA or with 5µl of 10 mM Tris-HCl (control) on ice for 5 min.
?. Put cells into a 0.2 cm cuvette, mix well with finger, electroporate using SHS (2.0 kV, 1 pulse), SC2 (1.5 kV, 1 pulse), or DIC (1.0 kV, 2 pulses, 1.0 msec) program with Bio-Rad MicroPulser 165-2100.
?. Add 1mL of L1 medium with antibiotics to the 0.2 cm cuvette, mix well, and transfer to a 1.5 mL tube, mix well and separate to different wells of a 12-well plate (with BASTA and without BASTA), add additional L1 medium with antibiotics to a total volume of 2 mL.
?. Incubate the 12 well plate in 25°C for F. kawagutii  and 15°C for A. catenella for 24 hours.
?. Add BASTA to the final concentration of 0.5-0.67 mg/ml for F. kawagutii and 0.07-0.1 mg/ml for A. catenella.
?. Observe cells under normal and epifluorescent microscope in 1-3 days and continue to monitor for several weeks.
?. Plasmids designed and used for F. kawagutii and A. catenella were DinoIII-bsr, DinoIII-pat, the previously described DinoIII-gfp (Sprecher et al., 2019), and vectors used in previous studies (Ortiz-Matamoros et al., 2015a; Ortiz-Matamoros et al., 2015b)
TITLE: qPCR assay for Aquarickettsia spp.
AUTHORS: Sterling R Butler, Stephanie Rosales

[DESCRIPTION]
qPCR for the quantification of Aquarickettsia spp. (Klinges et al., 2022) a putative parasite found in the coral A. cervicornis. This protocol has been altered by incorporating a recently published A. cervicornis CAM control gene (Palacio-Castro et al., 2021) targeted to detect differences across A. cervicornis genotypes because it is a single-copy gene in A. cervicornis.




[GUIDELINES]
PrimeTime MM - (keep in -20 for long storage) 
Forward coral host primer (Acropora) at 10 uM (keep in -20 for long storage) - Primer sequences from https://doi.org/10.1007/s00338-021-02159-x
Reverse coral host primer (Acropora) at 10 uM (keep in -20 for long storage) - Primer sequences from https://doi.org/10.1007/s00338-021-02159-x
Forward Aquarickettsia primer at 10 uM (keep in -20 for long storage) - Primer sequences from
Reverse Aquarickettsia primer at 10 uM (keep in -20 for long storage) - Primer sequences from

[STEPS]
SECTION: Prepare for qPCR
1. Remove PCR reagents from freezer and allow reagents to thaw on ice or at room temperature.
Wipe down PCR hood with 10% bleach and ethanol. 
Place consumables such as tubes, plates, plate sealers, and water in PCR hood and turn on UV light for 20 min 
Once everything is thawed vortex PCR reagents, spin them down, and place them on ice.
Keep reagents cool or on ice during the duration of the protocol. 

SECTION: qPCR thermocyler program settings
4. Select SYBER green and long run
 

SECTION: Prepare PCR master mix
2. Prepare enough master mix for the number of reactions needed. Each combination of sample and target (gene) should be run at least in duplicates. Add a few reactions to your calculations to account for pipetting errors.

A. cervicornis (CAM) master mix


 



A. rohwerii (tlc1) master mix
 
Combine all the PCR master-mix reagents in a microcentrifuge tube
Mix gently and spin down to collect mixture and remove bubbles
SECTION: Setup the qPCR plate
3. Add 8 uL of master mix to each well. Aiming for the bottom of the well will help to visualize what wells had master mix and DNA added.  
Add DNA to each well (2 uL). Aiming for the top of the well will help to visualize what wells had master mix and DNA added. 
Close the plate with optical clear caps or seals
Spin down the plate to mix the DNA and mastermix. 
Place in the qPCR machine and start the machine using the specified settings.
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Detection of Sars-Cov2 Using qPCR
AUTHORS: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">PCR based tests, such as quantitative PCR (qPCR) are the gold standard to test for the novel coronavirus (SARS-Cov2). Many labs regularly use qPCR for their own specific projects. The ability to process samples and detect SARS-Cov2 is not very different from the methods these labs are already intimately familiar with. This protocol outlines the methods that our group is using to detect SARS-Cov2 RNA in human samples. </div></div>

[STEPS]
?. [cDNA Synthesis]
Add 4 µL of Superscript IV VILO Master Mix (Fisher, 11-755-250) to each PCR tube (using PCR strip tubes).
?. [cDNA Synthesis]
Add 16ul RNA of RNA to each PCR tube (using PCR strip tubes).
?. [cDNA Synthesis]
Gently mix by inversion and briefly centrifuge to collect the liquid at the bottom of the tube.
?. [cDNA Synthesis]
Perform cDNA synthesis reaction in a Thermocycler (~30 min. total). •Constant lid temperature of 105ºC. •10 min. at 25ºC. •60 min. at 42ºC. •5 min. 85ºC. • Hold at 4ºC.
?. [qPCR]
Prepare master mix (written per reaction)  •10 µL 2x Taqman GEX Master Mix (Fisher, 4369016). •1 µL 20x Gene Expression Assay Probe. •3 µL DNase/RNase free molecular grade water.
?. [qPCR]
Dispense 14 µL of master mix into wells of a white semi-skirted PCR plate (USA Scientific, 1402-9790).
?. [qPCR]
Dispense 6 µL of cDNA into wells of a white semi-skirted PCR plate (USA Scientific, 1402-9790).
?. [qPCR]
Seal plate with optical plate sealing film (USA Scientific, 2978-2700).
?. [qPCR]
A human RNaseP, SARS-Cov2 N1, or SARS-Cov2 N2 probe is used in each reaction.
?. [qPCR]
Transfer plate to a real-time thermocycler. •2 min. at 50ºC •10 min. at 95ºC •40 cycles (15 s 95ºC, 1 min. 60ºC)
?. [Analysis]
Based on CDC guidelines, only CT values under 35 are valid for the test.
TITLE: Immunization with alpha-synuclein peptide
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details immunization of MHC-II(-/-); HLA DRB1*15:01 mice with alpha-synuclein(32-46) peptide in complete Freund's adjuvant.

[STEPS]
SECTION: Immunization
1. Prepare immunization emulsions:
SECTION: Immunization
1.1. Prepare 200 µg of M. tuberculosis H37Ra (BD Difco Adjuvant, Fisher Scientific, Cat #DF0638-60-7; Waltham, MA) in 1x PBS or in 100 µg a-syn32-46 peptide (A&A peptides; San Diego, CA) dissolved in 1x PBS.
SECTION: Immunization
2. Immunize 8- to 12-week-old male and female mice subcutaneously with 50 µL of emulsion into the chest area of the mouse, near the axillary lymph node at both front limbs for a totally of 100 µL of emulsion injected per mouse.

SECTION: Immunization
3. The day of immunization is designated as day 0.
SECTION: Immunization
4. At day 14, boost each mouse with the same immunization dose at the same site.
SECTION: Immunization
5. Administer intravenous injections of 400 ng of B. pertussis toxin (List Biological Laboratories, Cat #181; Campbell, CA) at 14- and 16-days post-initial immunization (day 0 and 2 post boost).

SECTION: Immunization
6. Monitor mice daily for their weight, food and fluid intake and neurological deficits and their weights are recorded daily. Mice are classified as sick if they displayed 12% reduction from 0 DPI weight and/or hunched posture with ruffled and ungroomed fur.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">This protocol is part of the manuscript: <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a><br /></span></p>
<p> </p>

[BEFORE_START]
<p>All reagents (1x PBS) and materials (pestles, beads) used in this protocol need to be sterile.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is modified from: </p>
<p>Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct<br />detection and enumeration of the fungilike marine protists, the thraustochytrids. Limnol.<br />Oceanogr. 38(1): 182-187.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a generic&nbsp;PNGase F protocol with non-denaturing reaction conditions. It is appropriate for both&nbsp;<a href="https://www.neb.com/products/p0704-pngase-f" target="_blank">P0704</a>&nbsp;and&nbsp;<a href="https://www.neb.com/products/p0708-pngase-f-recombinant" target="_blank">P0708</a>.&nbsp;Typical reaction conditions are below.

[BEFORE_START]
When deglycosylating a native glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the <a href="https://www.protocols.io/view/PNGase-F-Denaturing-Conditions-imsvtm" target="_blank">denaturing protocol</a> to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.

[GUIDELINES]
When deglycosylating a native glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the&nbsp;<a href='https://www.protocols.io/view/PNGase-F-Denaturing-Conditions-imsvtm' target='_blank'>denaturing protocol</a>&nbsp;to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.

[STEPS]
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TITLE: Measurement of GCase activity in lysosomes in live cells
AUTHORS: rachel.bates

[DESCRIPTION]


Assay uses the substrate PFB-FDGluc (5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside)from
ThermoScientific (Cat# P11947). Substrate is supposed to be taken up by late
endosomes and lysosomes only and fluoresces green when cleaved by lysosomal GCase.
Described by Mazzulli et al., J
Neurosci. 2016 Jul
20;36(29):7693-706. 




[STEPS]
4. Wash cells once carefully with prewarmed PBS.
6. Add 125ul substrate (400ug/ml substrate (400 mg/ml in OPTIMEM) per well. Put in 37 °C incubator for 1 h.
7. Aspirate substrate. Wash cells three times with 250 ml PBS (37 °C).
8. ADD 125UL ml OPTIMEM (37 °C). Measure t=0 minutes on plate reader: Ex, 488nm, Em, 520 nm. 
Return cells to incubator.

10. Optional: At end of experiment carefully aspirate OPTIMEM. Lyse cells in wells with 200 ml 1% TX-100 in PBS on ice for 15 minutes. Pellet debris at 17,000 xg, 10 minutes, 4°C. Measure protein concentration in supernatant with BCA assay. 
2. Culture cells in 48 well plate. Should be over 70% confluent to ensure a robust signal. If too confluent activity will plateau rapidly. 
1. Resuspend 5mg of substrate in 100 ul methanol to get 50mg/mL stock. Do not expose to light. Store at -20C
3. For control wells add 10uM CBE or 100uM Bafilomycin overnight. 
5. Working substrate solution is 400 mg/ml (I don’t get signal with lower
concentrations). Prepare in OPTIMEM prewarmed to 37 °C: 125 ml/well.
Therefore 1 ul of 50mg/ml stock substrate per 125 ml.
11. 
9.  Measure fluorescence every 20-30 minutes in green channel (excitation/emission maxima ∼492/516 nm) for up to 3 hours. Keep checking cells are alive with microscope.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Daniel Richter, Nov 29, 2011</p>
<p>based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F</p>

[GUIDELINES]
<p>To avoid possible RNA degradation, try to work quickly in all steps.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Procedure for the transformation of the nuclear genome of <em>Tetraselmis striata</em> by microprojectile bombardment.</p>

[GUIDELINES]
<p>The overall bombardment protocol has been modified from that used for chloroplast transformation of <em>Chlamydomonas reinhardtii</em> (Boynton and Gillham, 1993).</p>
<p> </p>
<p><strong>References </strong></p>
<p>Boynton J. E. and Gillham N. W. (1993) Chloroplast transformation in <em>Chlamydomonas</em>. <em>Meth. Enzymol.</em> 217: 510-536.</p>
<p> </p>
<p>Guillard R. R. L. and Ryther J. H. (1962) Studies of marine planktonic diatoms. I. <em>Cyclotella nana </em>Hustedt and <em>Detonula confervacea</em> Cleve. <em>Can. J. Microbiol.</em> 8: 229-239.</p>

[STEPS]
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TITLE: Feline Enteric Virus Detection Assay
AUTHORS: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

[DESCRIPTION]
The Feline Enteric Virus Detection Assay is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of feline coronavirus (FCoV)/feline infectious peritonitis (FIP), feline panleukopenia virus (FPV; feline parvovirus), feline rotavirus A (FRV) and SARS-CoV-2 in rectal swabs and fecal samples.

[GUIDELINES]
 SHIPPING & STORAGE INFORMATION
The Feline Enteric Virus Detection Assay is shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at -80°C.


LIMITATIONS:
Strict compliance with the instructions is required for optimal results.
Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
The presence of RT-PCR inhibitors may cause false negatives.
Results of Feline Enteric Viruses Detection Assay need to be interpreted in consideration of all clinical and laboratory findings.

QUALITY CONTROL:
The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA or plasmid copies number. All assays have a limit of detection (LOD95)  35 copies/l.

[STEPS]
SECTION: Reaction Set Up
1. Thaw all reagents on ice.
SECTION: Reaction Set Up
2. Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice 
 

SECTION: Reaction Set Up
3. Setup the Master Mix according to the following table 1:

  

 
 

SECTION: PROGRAMMING THE THERMOCYCLER
4. Select the following fluorescence channels: ABYTM, FAMTM, JUNTM and VICTM.
 

SECTION: PROGRAMMING THE THERMOCYCLER
5. The standard mode should be selected, following the table below:
 

SECTION: RESULTS INTERPRETATION
6. Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.


SECTION: RESULTS INTERPRETATION
7. Each assay is considered valid if the following criteria are met:
   

SECTION: RESULTS INTERPRETATION
8. The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To separate and visualize DNA fragments of varying sizes, using a gel matrix and an electrical current.</p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: Protein extraction form Aurantiochytrium limacinum (ATCC MYA-1381)
AUTHORS: Anbarasu Karthikaichamy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protein extraction protocol for </span><span style = "font-style:italic;">Aurantiochytrium limacinum</span><span> (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid). </span></div></div>

[STEPS]
?. [Lysis buffer preparation]
Start by making stocks of the following,  KCl MgCl2TrisPrepare lysis buffer (LyB) by adding the following chemicals (for 10ml),  AB1ChemicalVolume (uL)2KCl (1M)5003MgCl2 (25mM)10004Tris (1M)5005NP40 (100%)456Tween (100%)457dH2O8310
AB1ChemicalVolume (uL)2KCl (1M)5003MgCl2 (25mM)10004Tris (1M)5005NP40 (100%)456Tween (100%)457dH2O8310
?. [Cell harvesting]
Pipette out ~3 ml of cell suspension from the test tube or conica flask. Centrifuge the cells at   Discard the supernatant and keep the cell pellet on ice.Centrifuge the cells at
Centrifuge: 3000 33, 10 min, 4 10
?. [Lysis and soluble fraction extraction]
Add 1 ml of lysis buffe (LyB) to the pellet.Vortex the mixture for 20 mins on high spped.Centrifuge the suspension at    Collect the supernatant in a separate tube and place it on ice. This is the soluble fraction.Centrifuge the suspension at
Centrifuge: 13500 33, 15 min, 4 10
?. [Insoluble fraction extraction]
Resuspend the pellet from the above step in 4x LDS sample bufferAdd required amount of dH2OAdd 1:10(v:v) 25% β-Mercaptoethanol  Heat the mixture at    for   Centrifuge the mixture at   Carefully aspirate the supernatant (insoluble fraction) into a fresh tube and keep it on ice. Heat the mixture at   for Centrifuge the mixture at
Always handle β-Mercaptoethanol in fume hood!
70 °C
Centrifuge: 13500 33, 20 min, 4 10
Alternatively, one could also use custom buffers (with high concentration of detergent) for insoluble fraction extraction.
?. [Quantification and visualization]
Quantify the protein extract using an appropriate mehtod and the proteins can be visualized on a SDS-PAGE gel stained with coomassie blue.