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

[DESCRIPTION]
<p>Totally digest marine particles collected on polyethersulfone (PES, Pall Supor) filters.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the extraction process of RNA and/or DNA from marine environmental samples. Samples are assumed to be collected via filtering sample seawater onto filters. Filters are then flash frozen in the first lysis buffer. The protocol describes the steps used to lyse and separate material from filters and use the lysate for RNA and/or DNA extraction using Qiagen extraction kits (e.g. https://www.qiagen.com/us/shop/Sample-Technologies/Combined-Sample-Technologies/).</p>
<p> Downstream RNA and DNA is used for molecular analyses such as tag sequencing (16S or 18S), (meta)genomic, or (meta)transcriptomic applications.<br /><br />List of materials needed before starting is in <strong>Guidelines.</strong></p>

[BEFORE_START]
- Please read through all of the warnings, steps, and considerations for Qiagen extraction kit<br />- Ensure that Buffer RLT+ (or RLT) has beta mercaptoethanol added, as per the Qiagen instructions

[GUIDELINES]
<p>These steps for RNA (&amp; DNA) extraction are specific to samples of seawater filtered onto GF/F, nitex mesh, or polycarbonate filters*. This protocol focuses on additional steps taken for lysis and collection of material from filters before using a Qiagen DNA/RNA Allprep kit.</p>
<p> </p>
<p>Considerations:</p>
<ul>
<li>*Please confirm/test that protocol chemistry is compatible with filter of your choice. The beginning of this protocol assumes that collected filters were flash frozen into RLT+ Buffer (Qiagen)</li>
<li>This protocol is designed to also be compatible with other Qiagen extraction protocols. Please read all steps of the protocol ahead of time to confirm other Qiagen components are interchangeable.
<ul>
<li>Ensure loading material is within the range of extraction kit</li>
</ul>
</li>
<li>When working with RNA please minimize any exposure to non-RNase DNase safe environments and prior to lysing, ensure samples remain frozen at -80C.</li>
</ul>
<p> </p>
<p><strong>Materials</strong>:</p>
<ul>
<li>Qiagen extraction kit, can be from a variety of kits</li>
<li>RNase-free DNase set from Qiagen (cat # 79254)</li>
<li>Ice</li>
<li>Heat block (65C)</li>
<li>RNase-DNase clean pipets and filter pipet tips</li>
<li>gloves and RNase/DNase-free bench space</li>
<li>5mL sterile syringe, 1 needed for each filter</li>
<li>5mL centrifuge tubes, RNase-DNase free (additional UV-sterilization preferred)</li>
<li>5 mm Silica beads (e.g. Biospec products), made RNase-DNase free. Beads can be baked in oven to make RNase/DNase-free for 2 hrs at 550F.</li>
<li>vortex</li>
<li>Microcentrifuge, both desktop mini and larger centrifuge to fit 1.5mL tubes</li>
<li>96-100% EtOH</li>
<li>70% EtOH (Freshly made in sterile water)</li>
<li>Sterile forceps (or disposable forceps)</li>
</ul>

[STEPS]
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TITLE: Freezing cancer cell lines
AUTHORS: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines routine banking of cancer cell lines and Ca9 transduced cancer lines.</div><div class = "text-block">Process diagram:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/ccg3bdsax.png" /></div></div>

[STEPS]
?. Prepare   freezing media per vial as follows: complete culture media + 10% DMSO.
1 mL
?. Detach and collect cells from a flask, by following Steps 1-6 of the protocol: Passaging adherent cancer cell lines.
?. Aspirate the supernatant, taking care to avoid disturbing the cell pellet. Resuspend the pellet in an appropriate volume of freezing media- depending on the number of vials being frozen. Mix well to ensure a single cell suspension.For example, if 5 vials are being frozen from a T150, resuspend the cell pellet in  of freezing media.
5 mL
?. Transfer   aliquots of the cell suspension to pre-labelled 1.8ml cryovials.
1 mL
?. Place vials in a 'CoolCell' or appropriate freezing container and store at   overnight.
-80 °C
Appropriate freezing containers will ensure that the liquid freezes at a controlled rate of around   per minute at   .
?. Transfer the vials to liquid nitrogen for long-term storage.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Appropriate tissue fixation is essential for a good quality immunocytochemistry (ICC).</p>
<p> </p>
<p>There are several fixation methods, but "whole body" or "target" perfusion of the animal is one of the most efficient methods.</p>
<p>Most perfusing protocols include only paraformaldehyde, but acrolein penetrates tissue more rapidly, improving final fixation of the tissue.  </p>
<p> </p>

[STEPS]
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TITLE: Protocol Publishing QC Checklist (Warinner Group)
AUTHORS: James A Fellows Yates, Christina Warinner

[DESCRIPTION]
This protocol serves as a QC checklist which must be completed prior for a protocol to be made public, for all protocols written by members of the Warinner Group.

[BEFORE_START]
Use this checklist with the 'RUN' functionality of protocols.io. This can be accessed with the 'RUN' button to the left of share, when looking at the protocol in the file manager. This CAN be resumed, if not completed immediately.

 
 
Rename the document: '<YOUR PROTOCOL>  - QC Checklist'

Move the 'run' document in folder in the file manager under: Groups/WarinnerGroup/Protocol Publishing Checklist/Checklists.
 


[STEPS]
SECTION: Standardisation
1. Have all editors of the protocol been added as authors?
 

 

SECTION: Standardisation
2. Has the 'owner' of the protocol been transferred either to a main author or administrator (Tina, Irina, or Raphaela)?
SECTION: Standardisation
3. If this protocol has been previously published in a journal, has the citation been added under 'description'?
SECTION: Reagents
4. Are all materials, reagents and machines/devices added to materials?
 

SECTION: Reagents
5. Are all reagents the version that you used? If the kit has changed (e.g. MoBio kit is now renamed as PowerSoil), make sure that all future versions of the protocol have the latest version. If necessary, add this to the protocols.io database with SKU number and manufactuerers website link!
 

SECTION: Standardisation
8. Are all measurement units in a _universal_  format (i.e. they should be metric, or device type netural - RCF and not RPM for centrifgation)?
SECTION: Language
11. Optional if not a native speaker, have you had the protocol reviewed by a native speaker?
SECTION: Language
10. Has your language been reviewed for 'standard' protocol writing (i.e. as would be in a journal?) by someone experienced in the lab?
SECTION: Approval
14. Have you had your protocol approved by an administrator (Raphaela, Irina)?
SECTION: Approval
15. Have you had your protocol approved by the lab head (Tina)?
SECTION: Standardisation
6. Are all pre-preparation steps already defined early in the protocol (e.g. cooling a rack to 4 10 )?

SECTION: Standardisation
7. Have you indicated pause-points for all tube labelling steps (as this can take a long time)? 
SECTION: Language
12. Have you had the protocol reviewed by someone not familiar with the protocol or lab (to ensure there is no assumed language) 
SECTION: Publication Plan
13. Have you discussed with the lab whether this protocol should go through a 'public beta' phase (i.e. open for a fixed period for public comments prior publishing with a DOI)?
SECTION: Standardisation
9. Have all components been added, where applicable (i.e. amounts, temperatures, duration)
TITLE: OPestTL V1.1 -- the Open Pesticide Transition Library
AUTHORS: Benjamin  Orsburn

[DESCRIPTION]
Pesticide residue screening is a critical method for environmental and food safety. Today, disjointed libraries of pesticide transitions can make it challenging to develop new methods between instrument vendors. From the combined experience of thousands of  separate pesticide screening assays performed by this author, I have compiled a library of over 4,000 pesticide transitions for various instruments. These transitions are presented here in the form of the Open Pesticide Transition Library (OPestTL) along with optimized transitions and settings for insturments from 5 separate instrument vendors. Exact masses are also provided for screening with high resolution instruments in the form of the 3 most common adducts for these compounds. While a growing work, PestTL represents the single largest open library of pesticide transitions assembled to date. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p><span style="font-family: 'Segoe UI', sans-serif; font-size: small;">Adapted from Arnow, L. E. Colorimetric determination of components of 3,4-dihydroxyphenylalanine-tyrosine mixtures. </span><span style="font-family: 'Segoe UI', sans-serif; font-size: small;"><em>J Biol Chem</em></span> <span style="font-family: 'Segoe UI', sans-serif; font-size: small;"><strong>118</strong></span><span style="font-family: 'Segoe UI', sans-serif; font-size: small;">, 531-537 (1937).</span></p>

[STEPS]
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TITLE: DNA - Ball Python DNA Extraction from sheds
AUTHORS: Jose Avila Cervantes

[DESCRIPTION]
Protocol to extract DNA from Ball Python (Python regius) dry sheds using Phenol:Chloroform:Isoamyl Alcohol

[STEPS]
1. EQUIPMENT
Dry Bath / Heated Block
Microcentrifuge
DNA LoBind tubes 1.5mL
Micropipettes
Assorted pipette tips 
2. REAGENTS

Lysis Buffer (10 millimolar (mM)  Tris-base,  100 millimolar (mM)  EDTA, 2% SDS, 5 Molarity (M), NaCl, pH 8 )
TE Buffer (EDTA 1 millimolar (mM), Tris-Cl 10 millimolar (mM)  )
Proteinase K  (20 mg/mL ) 
Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v)
Ethanol 100 %
Ethanol 70 % (Freshly prepared)
Tris Acetate-EDTA (TAE) Buffer 
SYBR Safe DNA stain
Agarose
Loading Dye
Wide Range Ladder (100-12,000 bp)


3. PROTEINASE K DIGESTION

1. Cut a small piece of fresh tissue (3-5mm). Put in a 1,5mL microtube. Add 900 µL of lysis buffer. Add 9 µL  Proteinase K (20 mg/mL ) and vortex thoroughly for 30 s  seconds.

2. Incubate at 55 °C .


4. PHENOL/CHLOROFORM/ISOAMYL EXTRACTION
1. Add 500 µL of Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v) and vortex thoroughly for 30 s  seconds.

2. Centrifuge at room temperature for 5 min  at 16000 x g .

3. Carefully remove the upper aqueous phase (~500 µL )and transfer the layer to a fresh tube. Be sure not to carry over any Phenol during pipetting.
  

5. ETHANOL PRECIPITATION

1. Add 1000 µL of 100 % volume  Ethanol, invert the tube and place the tube at -80 °C for 60 min or at -20 °C   .

2. Centrifuge the sample at 4 °C for 30 min at 16000 x g to pellet the DNA.

3. Carefully remove the supernatant without disturbing the pellet.

4. Add 150 µL of 70 % volume ethanol.

5. Centrifuge the sample at 4 °C for 5 min at 16000 x g . 

6. Carefully remove the supernatant without disturbing the pellet.

7. Repeat 70 % volume  ethanol wash once and remove as much of the remaining ethanol as possible.

8. Dry the DNA at Room temperature for 5 min to 10 min .

9. Re-suspend the DNA pellet in 200 µL of TE Buffer.

6. DNA QUALITY CHECK

1. Prepare a 1% agarose gel with 1X TAE buffer and 1X SYBR Safe DNA stain.

2.  Add  4 µL  Wide Range Ladder (100-12,000 bp) in the first well.
 
3. Premix 9 µL of sample and 1 µL of 10X loading dye or 5 µL of sample and 1 µL of 6X loading dye. Load this mixture in each well.

3. Run the samples for 40 min at a 100V.

4. Visualize in a trans illuminator and take a photo of the gel.
TITLE: MSD V-PLEX Proinflammatory panel
AUTHORS: patricia.garcia

[DESCRIPTION]
The V-PLEX panel offers analytically validated multiplex assay kits. Developed under rigorous design control, V-PLEX kits provide accurate and reproducible results with consistency from lot to lot. The V-PLEX Proinflammatory Panel 1 (human) Kit includes ten biomarkers that are associated with the inflammatory response and immune system regulation.

[STEPS]
SECTION: MSD V-PLEX Proinflammatory Panel
1. Prepare calibrator dilutions
a.    Prepare the highest calibrator (Calibrator 1) by adding 1000mL of Diluent 2 to the lyophilized calibrator vial. After reconstituting, invert at least 3 times. Let it equilibrate for 15-30 minutes at room temperature and then vortex briefly using short pulses.
b.    Prepare the next calibrator by transferring 100mL to the highest calibrator to 300mL of Diluent 2.
c.    Use Diluent 2 as the zero calibrator.
SECTION: MSD V-PLEX Proinflammatory Panel
2. Prepare Detection Antibody Solution
For one plate, combine 2.400mL of Diluent 3 with:
-      60mL of SULFO-TAG Anti-hu IFN-γ Antibody
-      60mL of SULFO-TAG Anti-hu IL-1b Antibody
-      60mL of SULFO-TAG Anti-hu IL-2 Antibody
-      60mL of SULFO-TAG Anti-hu IL-4 Antibody
-      60mL of SULFO-TAG Anti-hu IL-6 Antibody
-      60mL of SULFO-TAG Anti-hu IL-8 Antibody
-      60mL of SULFO-TAG Anti-hu IL-10 Antibody
-      60mL of SULFO-TAG Anti-hu IL-12p70 Antibody
-      60mL of SULFO-TAG Anti-hu IL-13 Antibody
-      60mL of SULFO-TAG Anti-hu TNF-α Antibody
3. Prepare Wash Buffer
For one plate combine:
- 15mL of MSD Wash Buffer (20X)
- 285mL of deionized water
4. Prepare Read Buffer T
For one plate combine:
- 10mL of Read Buffer T (4X)
- 10mL of deionized water
5. Assay Protocol


5.1. Wash and Add Sample
a.    Wash the plate 3 times with at least 150mL/well of Wash Buffer.
b.    Add 50uL of prepared samples, calibrators, or controls per well. 
c.    Seal the plate with an adhesive plate seal.
d.    Incubate at room temperature with shaking for 2h.
5.2. Wash and Add Detection Antibody Solution
a.    Wash the plate 3 times with at least 150mL/well of Wash Buffer.
b.    Add 25uL of detection antibody solution to each well.
c.    Seal the plate with an adhesive plate seal.
d.    Incubate at room temperature with shaking for 2h.
5.3. Wash and Read
a.    Wash the plate 3 times with at least 150mL/well of Wash Buffer.
b.    Add 150uL of 2X Read Buffer T to each well.
c.    Seal the plate with an adhesive plate seal.
d.    Analyze the plate on an MSD instrument.
TITLE: Preparation of PEI for primary coating of MaxOne (neuronal cultures)
AUTHORS: Yonatan Katz

[DESCRIPTION]
Polyethyleneimine (PEI) coating to enhance cell adhesion on MaxOne chips. PEI coating is followed by Laminin coating.

[STEPS]
SECTION: 0.07% PEI solution preparation - Primary coating
2. Prepare 100 mL of 1X borate buffer: 
5 mL 
95 mL 

SECTION: 0.07% PEI solution preparation - Primary coating
3. 7% PEI stock solution: 
1.4 mL 
8.6 mL 

Mix 30 min  using a magnetic stirrer until fully dissolved
(can be stored in 0.5 mL aliquots at -20°C for 1 month).
Where is it sterilized? -  

SECTION: Materials
1.   
  

SECTION: 0.07% PEI solution preparation - Primary coating
4. final 0.07% PEI solution
0.5 mL in
49.5 mL 

filter through a 0.22 μm filter unit before use!
TITLE: Western Blot
AUTHORS: James Montgomery

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Gel Electrophoresis    ]
Make loading dye using LDS Sample Buffer (4X) and Reducing agent (10X). For   of sample, add  sample buffer and  reducing agent, keeping in mind that each well holds  total.
20 µl
7.5 µl
3.5 µl
30 µl
?. [Gel Electrophoresis    ]
Combine sample and loading dye and heat at   for  .
95 °C
?. [Gel Electrophoresis    ]
Prepare precast gel by removing comb and the tape at the bottom of the gel chamber, making sure to remove any excess gel that could block the top of the wells. Construct the gel chamber in the gel tank such that the well openings of the precast are facing inwards toward eachother. Fill the gel chamber with (1X) MOPS running buffer and  antioxidant.
200 mL
500 µl
?. [Gel Electrophoresis    ]
Remove samples from heat and cool  for  .
on ice
?. [Gel Electrophoresis    ]
Spin samples at 10K for
?. [Gel Electrophoresis    ]
Load samples into gel using hamilton pipette tips. Be sure to record the order of the samples in the gel noting that samples on the edges of the gel are more likely to become distorted. Placing the marker in the middle most gel will provide a more reliable size ladder for size estimation.
?. [Gel Electrophoresis    ]
Run gel at 100V for  or until the protein has migrated far enough into the gel.
?. [Transfer]
While the gel is running prepare    using  ,  , and   and place in cold room for duration of gel run.
[Transfer Buffer]
[DiH20]
[10x Transfer Buffer]
[methanol]
?. [Transfer]
Soak membrane (for PVDF soak membrane in methanol, for nitrocellulose soak membrane in transfer buffer.) Do not touch membrane with ungloved hands.    Soak sandwhich components (2 sheets of filter paper and 2 pads) until completely damp. Make sandwhich being careful to avoid trapping any air between layers. Place cassette in transfer tank with transfer buffer and run for 1 hour.
?. [Blocking]
Remove membrane from sandwhich, noting the orientation relative to the gel. Wash the membrane in 1X PBST for
?. [Blocking]
Block membrane for   with 5% milk in PBST
?. [Blocking]
Blot with desired Ab.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A protocol to separate chloroplasts from diatom cells using ammonium fluoride to permeate the silica frustrule and a percoll gradient to separate the plastid from other cellular components.

[BEFORE_START]
Make sure all buffers are prepared according to the instructions in "Buffers for Chloroplast Isolation from Diatoms"

[GUIDELINES]
All steps should be done with cold reagents and samples should be placed on ice between steps.<br />Buffers and reagents can be mixed up ahead of time and stored cold (refrigerated). Percoll gradients (layering of 40% and 80% percoll solutions) should be done the same day of the extraction shortly before use.

[STEPS]
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TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>This NSF (National Science Foundation) generic template includes the following sections: </p>
<p> </p>
<p>1. Types of data produced</p>
<p>2. Data and metadata standards</p>
<p>3. Policies for access and sharing</p>
<p>4. Policies for re-use, re-distribution, derivatives</p>
<p>5. Plans for archiving and preservation</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>RefSeqMash uses the MinHash (http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0997-x) algorithm to characterize the genera/species/strains present in metagenomic samples by comparing the reads to ~11K known RefSeq genomes.</p>

[STEPS]
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TITLE: Flow Cytometry ICS Nuclear Antigens
AUTHORS: Michael Betts, Gregory Golden

[DESCRIPTION]
High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. A combined analysis of extracellular epitope staining (ECS) and intra-cellular epitope staining (ICS) using a collection of fluorophore-labeled antibodies sufficiently identifies discrete cell populations and their respective phenotypes. Importantly, ECS/ICS can be applied to cryo-preserved cell suspensions recovered in tissue culture media, enabling samples to be conveniently analyzed after collection and proper cryopreservation. However, consistent cryopreserved sample recovery and ECS procedures are critical to data comparison across multiple experiments. Herein, we describe a standardized protocol for cryopreserved sample recovery and ECS/ICS procedures for cell-surface and intra-cellular epitope labeling.

[STEPS]
SECTION: Procedure
1. Thawing and Resting

a. Pre-warm R10 media in a 37 °C water bath. 

b. Thaw samples in-vial using a 37 °C water bath.

c. Add thawed cells to 14 mL of R10, then spin cells at 500 xg for 5 min.

d. Resuspend cell pellet in 3 mLof R10 and count cells. 

e. Rest cells at least 3 hours (up to overnight) at 2x106 cells/mL in R10 medium + 1 µL/mL DNAse I at 37 °C , 5% CO².
Note: during resting, prepare antibody cocktail master mix. Adjust volume of ECS for 50 µL per test with FACS buffer.

f. After resting, add PBS up to 15 mL or 50 mL (whichever is closer, rounding up) to cells and transfer to 15 mL or 50 mL conical tube.

g. Spin cells at 500 xg for 5 minutes at room temperature (RT).

h. Resuspend cells in PBS to 1x107 cells/mL and count. If cells are too dilute, re-spin cells and resuspend at 1x107 cells/mL. Transfer 200 mL of cells (2x106 cells) into each well of a V-bottom 96 well plate.

SECTION: Procedure
2. Viability and extracellular staining (ECS)

a. Spin plate at 500 xg for 5 minutes at RT

b. Using a multichannel pipette, carefully remove the supernatant. 

c. Add 5 µLof 1:60 Aqua viability dye directly to cell pellet and resuspend cells.

d. Incubate for 10 minutes at RT in the dark.

e. Add 50 µLof ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).

f. Add 100 µL of FACS buffer to each well and spin plate at 500 xg for 5 minutes. Remove supernatant. 

SECTION: Procedure
3. Fixation, Permeabilization, and ICS Staining

a. Fix cells with 100 µL of 1xFixation/permeabilization buffer (1 part concentrate + 3 parts diluent) for 30 minutes at RT in the dark.
Note: Make ICS abs here

b. Add 100 µLof 1x perm/wash buffer and centrifuge 800 xg for 5 min.

c. Remove supernatant and add 100 µLICS cocktail (made in perm/wash buffer, made with diH²O) to the cells.

d. Incubate for 1h at RT in the dark.

e. Add 100 µL perm/wash buffer. Centrifuge at 800 xg for 5 min.

f. Discard supernatant and resuspend pellet in 200 µL1% PFA and transfer to FACS tubes.

g. Wrap in aluminum foil and store at 4 °C until flow cytometry.
TITLE: Yale - Blood and Urine Inorganic Phosphorous
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of inorganic phosphorus in blood, serum, and plasma. Phosphorus (Inorganic) is measured as the complex with molybdate and monitored at 340nm.</div></div>

[STEPS]
?. Calibrate Cobas for Phosphorus analysis by running a multi-analyte serum calibrator and two control serum.
?. Sample handling as performed by Cobas Mira Plus     a) Pipette 3 µL of sample into cuvette.    b) Add 280 µL of Phosphorus Liquid Reagent.    c) Mixture is incubated at 37˚C for 10 minutes    d) Absorbance is measured at 340nm.