eamag/protocols-with-steps huggingface dataset:
TITLE: Generation of Flp-In™ T-REx™ 293 cells stably expressing VPS13C^mClover under a tetracycline inducible promoter
AUTHORS: Xinbo Wang, Shujun Cai, Will Hancock-Cerutti, Pietro De Camilli

[DESCRIPTION]
This method describes the generation of Flp-In™ T-REx™ 293 cells stably expressing internally mClover tagged VPS13C under a tetracycline inducible promoter.

[STEPS]
SECTION: Cloning
1. Clone gene of interest into pcDNA5/FRT/TO. For VPS13C^mClover the In-Fusion system (Takara Catalog # 638947) was used. 
SECTION: Cloning
2. Sequence plasmid.
SECTION: Cell culture
3. Thaw and culture Flp-In™ T-REx™ 293 cells according to Gibco manual (Catalog # R780-07) in a 10cm dish. Once cells are 90% Confluent, split.
SECTION: Cell culture
4. Freeze some cells according to Gibco manual protocol for future use.
SECTION: Transfection and selection of stably integrated cells
5. Plate cells in 6 well format to ~30% confluence in media containing no antibiotics.
SECTION: Transfection and selection of stably integrated cells
6. The next day, transfect cells using Fugene HD and a ratio of 9:1 pOG44: pcDNA5/FRT/TO-VPS13C^mClover.
SECTION: Transfection and selection of stably integrated cells
7. Mix 2 µg total DNA (0.2 µg pcDNA5/FRT/TO-VPS13C^mClover, 1.8 µg pOG44) in 100 µL opti-MEM.
SECTION: Transfection and selection of stably integrated cells
8. Add 8 µL Fugene HD to DNA mixture. Let sit 15 min.
SECTION: Transfection and selection of stably integrated cells
9. Add Fugene/DNA mixture to cells in a dropwise fashion.
SECTION: Transfection and selection of stably integrated cells
10. At 24h post transfection, wash cells and add fresh post-transfection media containing Blasticidin (no Zeocin or Hygromycin).
SECTION: Transfection and selection of stably integrated cells
11. At 48h post-transfection, split cells and plate at <25% confluence in selection medium containing Blasticidin and 200 μg/ml Hygromycin B (no zeocin).
SECTION: Transfection and selection of stably integrated cells
12. Feed the cells with Hygromycin B selection medium every 3-4 days.
SECTION: Transfection and selection of stably integrated cells
13. When foci become apparent, pool Foci and allow cells to expand.

 

SECTION: Transfection and selection of stably integrated cells
14. Pooled cells can then be split, frozen, and/or used for experiments.
SECTION: Transfection and selection of stably integrated cells
15. Freeze cells according to Gibco manual.
SECTION: Expression of VPS13C
16. Plate cells at desired confluence in format appropriate for experiment in expression media containing 0.1 μg/ml-1 μg/ml tetracycline.

 

SECTION: Expression of VPS13C
17. After 24 hours, cells may be lysed for western blot or used for microscopy.
TITLE: U Mass - Bilirubin
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of bilirubin reflect bile acid metabolism.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry
analyzer.
?. Select Bilirubin test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Total Health ACV Keto Gummies
AUTHORS: kzlwokvz  

[DESCRIPTION]
Total Health ACV Keto Gummies

[STEPS]
1. It can in like manner be trying to follow a low-carb diet plan, causing less accomplishment with a yo diet plan and keeping a sound weight. Assuming you attempt Total Health ACV Keto Gummies, you can bring down your gamble by picking lean meats, fish, and vegan protein sources, choosing low-fat dairy items, eating more green and lean vegetables, and eating all the more endlessly immersed fats. You can restrict your trans fat utilization. vegetables. Veggies with starch. On the off chance that you select a low-carb diet, you don't need to consider calories long as you keep your carb utilization low and remain with low-carb proteins, fats, and vegetables. Assuming that you cut calories, it doesn't mean you want to eat less.
https://www.facebook.com/totalhealthacvketogummies/
https://sites.google.com/view/total-health-acv-keto-gummies-/
https://www.scoop.it/topic/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted
https://techplanet.today/post/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted
https://lexcliq.com/total-health-acv-keto-gummies-pros-and-cons-is-it-scam-or-trusted/

2.
TITLE: Cell culture
AUTHORS: Alexander Röntgen

[DESCRIPTION]
Protocol to culture SH-SY5Y cells.

[STEPS]
SECTION: Cell culture
1. Culture human SH-SH5Y cells at 37 °C and 5% CO2
SECTION: Cell culture
2. Split cells at 80–90% confluency

SECTION: Cell culture
2.1. Discard medium, rinse with PBS
SECTION: Cell culture
2.2. Incubate cells with Trypsin-EDTA for 5 min at 37 °C and 5% CO2
SECTION: Cell culture
2.3. Add 9 mL medium to neutralise Trypsin-EDTA
SECTION: Cell culture
2.4. Centrifuge at 300 g, 5 min, RT
SECTION: Cell culture
2.5. Discard medium
SECTION: Cell culture
2.6. Resuspend in 10 mL medium
SECTION: Cell culture
3. Split at desired ratio into fresh medium
TITLE: Coding Manual for Childhood Obesity-Relevant Policy Documents
AUTHORS: Itzhak Yanovitzky, Matthew S. Weber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This is a new version of the protocol - after doing our study, we found that codes x through y didn't really help us much so we changed them in particular ways</span></div><div class = "text-block">•	Studies about policymakers’ use of research evidence frequently track ‘evidence’ as opposed to tracking ‘use’.        </div><div class = "text-block">•	Policymakers use evidence to inform their thoughts and decisions but also to influence those of others.</div><div class = "text-block">•	Policymakers’ communication with and about evidence is a reliable proxy of how and for what purpose they use evidence.</div><div class = "text-block">•	Methods from the field of persuasive communication can yield a rich and nuanced account of policymakers’ evidence use.</div><div class = "text-block">This is a protocol to code documents about the policymaking process using a theory-grounded content analysis methodology.  It looked at policymaking within U.S. Congress and the type and content of documents and evidence that was cited in deliberations</div></div>

[STEPS]
TITLE: Protocol for Generating Stably Expressed Mammalian Cell Lines Using Lentivirus
AUTHORS: creative-biogene

[DESCRIPTION]
Stable cell lines generated using lentivirus exhibit long term protein expression and the system is highly reproducible. In this protocol, we describe the use of the 3rd generation lentiviral system which uses three different plasmids for generating stable cell lines.

[STEPS]
SECTION: Experiment Summary
1. Stable cell lines generated using lentivirus exhibit long term protein expression and the system is highly reproducible. In this protocol, we describe the use of the 3rd generation lentiviral system which uses three different plasmids for generating stable cell lines. First plasmid contains your gene of interest usually flanked by Long Terminal Repeat (LTR) sequences, that are integrated into the host genome. Second is the pCMV Delta R8.2 plasmid that encodes all components necessary for packaging the lentivirus viz. HIV-1 Gag, Pol, Tat and Rev. Third and the final plasmid is the pCMV VSVG that encodes the viral envelope protein.
2. 
SECTION: Equipment
3. 1. Cell culture incubators (37 °C, 5% CO2and 32 °C, 5% CO2)
2. Hemocytometer (Standard)
3. Laminar hood BSL-2 type
4. 37 °C water bath
5. Autoclave
6. Micropipettes (1,000 μl, 200 μl)
7. -80 °C freezer
SECTION: Procedure
4. A. Preparation of lentivirus in HEK293T cells
1. Day 0: Seed about 2-3 million HEK293T cells in a 10 cm2plate.
2. Day 1: HEK293T cells should be about 70-80% confluent. If for some reason they seem less you may wait for another 8-10 h.
3. Prepare transfection mix using the following proportions in a sterile 1.5 ml Eppendorf tube as per manufacturer's instructions:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div class="b-start-block editable-content">
<p>GreenGlo ™, 20,000X in Water, is a non-carcinogenic and non-toxic alternative to Ethidium bromide used for the detection of nucleic acids in agarose gels. It is as sensitive as Ethidium bromide. There is no toxic DMSO as GreenGlo ™ is supplied in water. <br /> <br />GreenGlo ™ has fluorescence excitation maxima at 295 nm and 490 nm. The fluorescence emission maxima is similar to EtBr when bound to DNA – at 530 nm.</p>
<p> </p>
<p>Please follow one of the two protocols below, depending on whether you are pre-staining or post-staining.</p>
</div>
</div>
</div>
</div>

[STEPS]
TITLE: C4 ZipTip Solid Phase Extraction
AUTHORS: Lauren  Adams

[DESCRIPTION]
Solid phase extraction for clean-up and concentration of proteins prior to introduction into the mass spectrometer. 

[STEPS]
1. Activate Ziptip by pipetting 10 µL of C4 ZipTip Activation Buffer and discarding onto a Kimwipe for a total of 6 times.
2. Equilibrate the Ziptip by pipetting 10 µL of C4 ZipTip Equilibration/Wash Buffer and discarding onto a Kimwipe for a total of 6 times.

3. Remove C4 Ziptip from p20 pipette and place safely back into tip box to hold. Take a p200 pipette set at 200 µL and add a p200 pipette tip to the end. Take the p200 with pipette tip and then add the C4 ZipTip to the end. Carefully pipette the elution sample up and down for a total of 10 times per elution fraction. Avoid forcing air bubbles through the pipette tip as this will disrupt the resin and introduce oxidation to the target protein.
4. Remove C4 Ziptip from p200 pipette and place safely back into tip box to hold. Reattach to the p20 pipette and by pipet 10 µL of C4 ZipTip Equilibration/Wash Buffer and discard onto a Kimwipe for a total of 10 times.
5. Pipette into 5 µL of C4 ZipTip Elution Buffer that is inside a clean LoBind tube a total of 10 times. Dilute the final volume up to 25 µL for LC-MS or 80 µL for I2MS.
TITLE: Shipping protocols 
AUTHORS: Gabriel J J.  Barrero, Abraham  Palmer, Oksana  Polesskaya

[DESCRIPTION]
Shipping tissue protocol 

[STEPS]
SECTION: Collection
1. Collection
SECTION: Collection
1.1. Put the spleen into a pre-labeled 2 mLmicrocentrifuge tube (for example, Fisher Scientific cat#05-408-138). Add approximately0.5 mLsaline solution in the tube with the spleen to prevent dry-freezing of tissue, but make sure that the tube is not over-filled.
 If your rats have been implanted with an RFID chip, try to find this chip during dissection and put it directly into the tube with the spleen. We will read both the barcode and RFID chip to confirm the sample identity
. 
SECTION: Collection
1.2. For tube labels use “Cryo Babies” for laser printers (Diversified Biotech cat# LCRY-1700), they withstand long-term storage in -80 °Cwithout peeling off the tubes. 
    
 

SECTION: Collection
1.3. ·Store samples in cardboard boxes clearly labeled with the PI name, date (or at least year), tissue (e.g. “spleen”) and project title. Example:JHOU-HS ratsU01-Spleen-Box02_25Feb2019
  



SECTION: Collection
1.4. For a genotyping backup tissue sample, cut off ~ 5 mm of the tail tip and store at your lab. Use 2 mL tube labeled with the barcode and rat ID, add saline to prevent sample desiccation. Store at -80 °C long-term.

SECTION: shipping
2. Shipping
SECTION: shipping
2.1. Send spleens in the cardboard box (not plastic, they can break during shipping) labeled with the Project, type of tissue, box number, and the date (e.g. JHOU_HS rats-Spleen_Box02_25Feb2019). 
SECTION: shipping
2.2. Use a secondary container (plastic bag or just wrap with paper) in case box is broken during shipment. 
SECTION: shipping
2.3. Use at least 10 kg (20 lb) dry ice for shipping. If you have a choice, use pellets. If you use slabs, make sure that there is no free space in the shipping box, so slabs cannot move freely and crash things.
SECTION: shipping
2.4. Use Styrofoam box that is surrounded by a cardboard box. Choose boxes with thick walls. For example, ULINE S-13392 or ULINE S-11357. 
SECTION: shipping
2.5. ·Send by FedEx overnight on Monday or Tuesday, check for holidays. 
SECTION: shipping
2.6. At the time of shipping, send the manifest to the Center that contains a list of samples, box ID, collection date, and comments (if any), we will use it to update the database
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describe how to asess secretion efficiency of diferrent constructs in <em>Chalmydomonas reinhardtii</em>, using a fluorescent protein. Colonies are picked from transformation plate and inoculated on TAP media for growth. A high number of colonies is picked to avoid bias on colonie picking. </p>

[BEFORE_START]
<ul>
<li>Separate 96 well plates</li>
<li>Prepare sufficient media </li>
<li>Separate gas exchangable seal </li>
</ul>

[GUIDELINES]
<p>Cell cultures in 96 well plates should ideally be cultivated at high orbital shaking (&gt;800RPM) to avoid suboptimal growth. </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Protocols for &#34;Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death&#34;
AUTHORS: mai.nguyen, Jack Collier, Olesia Ignatenko, Sidong Huang, Michel Desjardins, heidi.mcbride

[DESCRIPTION]
Cell death is inhibited in cancers but increased in neurodegeneration, highlighting that its regulation is essential for human health. MAPL is an outer mitochondrial membrane SUMO ligase with roles in cancer and neurodegeneration in vivo, yet how MAPL controls cell death remains unclear. Combining genome-wide screening and cell biological approaches, we found that MAPL induces pyroptosis through an inflammatory pathway involving mitochondria and lysosomes. MAPL overexpression promotes mitochondrial DNA (mtDNA) trafficking in mitochondrial-derived vesicles (MDVs) to lysosomes, which are permeabilised in a process requiring gasdermin pores. This triggers release of mtDNA into cytosol, activating the DNA sensor cGAS, which is required for cell death. Additionally, multiple Parkinson’s disease-related genes, including VPS35 and LRRK2, also regulate MAPL-induced pyroptosis. Consistent with our findings, genetic deletion of MAPL, LRRK2 or VPS35 inhibited inflammatory cell death in primary macrophages, placing MAPL and the mitochondria to lysosome pathway at the nexus of immune signalling and cell death.  



[STEPS]
TITLE: Extraction of Oxycodone from Plasma for Mass Spec Analysis
AUTHORS: Sierra Simpson, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">From the Momper Lab.</div></div>

[STEPS]
?. [Harvesting Blood ]
Collect blood in a tube coated with EDTA. Heparin leads to hemolysis which can effect downstream processing.
?. [Harvesting Blood ]
Spin blood at 3000rpm for 10 minutes to separate erythrocytes from plasma
?. [Harvesting Blood ]
Aliquot plasma into 50-100ul aliquits to avoid freeze thaw. 20ul is processed per sample.
?. [Plasma Preparation]
Add 20ul plasma to 60ul Acetonitrile spiked with 500ng/ml Oxycodone-d-6 ( Heavy oxy for internal standard) This will crash out all the extra protein within the sample.
?. [Plasma Preparation]
Spin the sample at 13K for 10 minutes, then extract supernatant and put it into a mass spec vial for sampling.
?. [Running the samples ]
Load the samples into the autosampler 20µl supernatant is injected directly onto a C-18 reversed phase HPLC column (MacMod Ace-5, 2.1 x 150 mm).
?. [Running the samples ]
The LC mobile phase consisted of HPLC grade water with 0.1% formic acid (elute A) and ACN with 0.1% formic acid (elute B), and eluted with a gradient program of 0-.25 min / 95%B, flow rate of 0.4 mL/min; 1.00-3.00 min / 95 % B, flow rate of 0.6 mL/min;3.10-6.00 min / 5 % B, flow rate of 0.3 mL/min. MS/MS detection is made in positive electrospray ionization mode, at mass transitions of 316→241 m/z (Oxycodone), 322→247 m/z (ISTD).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Project Summary </strong></p>
<p><strong>Background Rationale </strong></p>
<p>Neonatal hypoglycaemia is a common condition, particularly affecting LBW newborns, and is associated with both acute and long standing complications. Ideally, the mother can provide breast milk as soon as possible; however, in many circumstances (poor nutrition of mother, post-operative Caesarian section, etc) this may not be the case for hours or even days. If resources are available, this can be managed by providing the LBW neonate with artificial milk/donor whilst waiting for breast milk when required. However, this is often not available in resource poor countries due to expense and difficulty in production (timely clean safe water, training in how to make the milk etc). Furthermore, in resourced hospitals, glucose is monitored in at risk babies, and should they develop hypoglycemia, IV 10% dextrose can be relatively simply administered. In resource poor settings, this is challenging- firstly, glucose monitoring and then the administration of IV 10% dextrose require resources, skills and nursing that are often not available. The primary aim of this study is to assess whether the use of oral 10% dextrose, is tolerable and reduces the incidence of hypoglycaemia in LBW newborns in the absence of sufficient maternal breast milk. 10% dextrose is readily available, inexpensive and can be stored at room temperature. </p>
<p><strong>Objectives </strong></p>
<p>Our aim was to assess whether hypoglycaemia could be prevented in LBW babies with insufficient maternal breast milk (MBM), by supplementing their feed with oral 10% dextrose. </p>
<p><strong>Methods </strong></p>
<p>This is a 8 months, interventional, prospective, single centre, randomised, double arm control trial on 74 newborns conducted at a district hospital in Tanzania (Tosamaganga Hospital) where Doctors with Africa - CUAMM has ongoing projects on maternal-neonatal health. LBW newborns fulfilling the inclusion criteria will be randomly allocated to either the intervention (supplemental oral 10% dextrose) or monitoring arm. Glucose will be monitored regularly in both arms and the incidence of hypoglycaemic episodes recorded together with normal vital signs. A budget of 16,000,000Tsh has been proposed for the study. </p>
<p><strong>Outcomes </strong></p>
<p>The primary outcome will be hypoglycemia (&lt;2·6mmol/L) in the first three days of life. </p>
<p> </p>

[STEPS]
?. 
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?. 
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?. 
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TITLE: 2100 Bioanalyzer Operation for PCR, GTP and IVT Products
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes bioanalyzer operation. The Agilent Bioanalyzer is used as a quality control step for PCR and IVT products.  Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Magnetic bead cleaning of PCR products with Cytiva Sera-Mag Select
AUTHORS: Alexander Eiler, Even Garvang

[DESCRIPTION]
Magnetic bead cleaning of PCR products, adapted for Cytiva Sera-Mag Select beads. Forked from an older protocol using AMPure XP beads with minimal changes.

[GUIDELINES]
Work at post-PCR lab.
You can use PCR tubes/strips or PCR-plates but for easiest work with magnetic beads flat-bottom 96-well microplates are recommended.

[STEPS]
1. Calculate the volume needed for the beads. For normal length primers a bead:sample ratio of 0.8:1 is likely OK. For long primers you can use a ratio of down to 0.65:1 (probably even lower). If you can still see primer dimers on a gel after cleanup, try a lower bead:sample ratio.

0.8 * sample volume =
2. Shake the magnetic bead buffer bottle to fully resuspend magnetic particles.
4. Add Sample.
5. Add magnetic beads. Pipette mix 10 times and Incubate at room temperature for 5 minutes.
5 min
6. Place the reaction plate onto a Magnet Plate for 2 minutes to separate beads from solution. (Keep on the magnet plate until step 11).
2 min
7. Aspirate the supernatant from the reaction plate and discard.
13. Add 40 μL of elution buffer (PCR water or 10mM Tris), pipette mix 10 times. Make sure the buffer reaches up to the beads!
40 1
15. Place the reaction plate onto a Magnet Plate for 1 minute to separate beads from solution. 
1 min
16. Transfer purified product to a new PCR plate or strips. Ensure that no beads are carried over when transferring, as it may inhibit later steps.
8. Dispense 200 μL of 85% ethanol and incubate at room temperature for at least 30 seconds. Aspirate out the ethanol and discard. You can discard the ethanol by inverting the plate keeping the magnetic plate on the bottom of it.
200 1
30 s
12. Dry off the remaining ethanol. To speed up the process, you can place the plate on a warm (30°C) heat block.
Drying takes at least 30 minutes. Check that drying was complete before continuing.
30 min
9. Repeat the washing step as above: dispense 200 μL of 85% ethanol and incubate at room temperature for at least 30 seconds. Aspirate out the ethanol and discard. You can discard the ethanol by inverting the plate keeping the magnetic plate on the bottom of it.
200 1
30 s
10. Remove the remaining ethanol by pipetting.
11. Take the plate off the magnet
14. Incubate at room temperature for 2 minutes.
2 min
3. Prepare 85% ethanol. You need 400 µl per sample, but prepare some extra. 

Remember that when mixing ethanol and water, the total volume decreases (e.g. 15 ml water + 85 ml 100% ethanol < 100ml 85% ethanol). For the same reason, measure ethanol and water separately before mixing
5. 
4.