eamag/protocols-with-steps huggingface dataset:
TITLE: Power Keto Gummies Reviews - Fake Side Effects Scam?
AUTHORS: keto-x-reviews  

[DESCRIPTION]
Power Keto Gummies Reviews - Fake Side Effects Scam?

[STEPS]
SECTION: Power Keto Gummies Reviews - Fake Side Effects Scam? Power Keto Gummies give colorful medical benefits to help those who can not bear the discomfort of taking colorful emulsion- loaded specifics to get well and end their regular straits. Salutary inconsistency and a lack of physical exertion are the two biggest pitfalls facing the average mortal.   When faced with this dilemma, the stylish course of action is to look for nearly any remedial option that can help homogenize clinical conditions while having no adverse side goods. The good news is that leathery delicacies like these are always ready to help. They claim that they&#x27;re entirely normal and set to produce the stylish results possible in the body.  Power Keto Gummies : What Are They?   This Keto sticky is a great way to lose weight and shape. Conventional diet and exercise rules have been reported to take up a considerable knob of the day to burn fat. So rather of devouring it with a revenge, our systems constantly store fat. That is each about to change, however. Start consuming fat in your foundation because your body&#x27;s turn is reversed in this composition. You will have further energy and a great mood as you get fitter.   Figure interpretations grounded on the typical sections of these Keto Gummies can be plant then.  They make you happy while also delivering better results. To fleetly test them out, cock on them like that! An inconceivable event is taking place. A long list of adverse issues eventually begins to crop. Make sure the results keep flowing in but do not allow them to get out of control. Keto Gummies use a unique approach to weight loss. The primary Keto Constituents determine whether or not a diet is complete, as preliminarily said.    Official Website:- https://ipsnews.net/business/2022/05/01/power-keto-gummies-reviews-consumer-scam-reports-read-now/
1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The description and identification of metabolites using Gas Chromatography - Mass Spectrometry (GC-MS) is a powerful tool to study the cnidarian-dinoflagellate symbiosis. The applications range from first descriptions of metabolite profiles to the identification of specific metabolic responses (e.g. biomarkers) of the cnidarian metaorganism (‘holobiont’), including its associated symbionts, under stress.</p>
<p> </p>
<p>The following step-by-step protocol is optimized to characterize the carbohydrate fraction from Aiptasia anemones or coral fragments, as well as their algal symbionts, using a targeted GC-MS approach. The protocol was previously used in Ochsenkühn et al. (2017) where it identified the following carbohydrates (floridoside, inositol, mannitol, glucose, glycerol, galactose, ribose and fructose) as well as some amino acids (glycine, alanine, valine and proline).</p>
<p> </p>
<p>In addition to the step-by-step protocol for sample preparation and derivatization, we provide detailed settings for the Agilent GC-MS system (GC (Agilent 7890A) and MS (Agilent 5975C)) in a separate document.</p>

[GUIDELINES]
<p>It is crucial that all steps, until the derivatization, are performed on ice to minimize degradation of metabolites. Normalization is essential for GC-MS data, as such, two options (dry-weight and total protein content) are included in this protocol.</p>

[STEPS]
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TITLE: Picking Clones
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Coat 96 well plate with 50 μl Matrigel per well.
?. Incubate at  for .
?. Prior to picking, aspirate Matrigel. Replace with  mTesR1 + 10 μM Rock Inhibitor.
50 µl
?. Using p20, pick individual clones and place into unique wells. Use a new tip with each clone.
?. Incubate at overnight.
?. Change media daily (150 μl mTesR1).
TITLE: Top2 inhibitor sensitivity
AUTHORS: Jacob Kirkland

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for Top2i sensitivity in attached cells in 96-well format. Short term exposure to Top2i (~3 days)</div></div>

[STEPS]
?. Plate (2) 6cm dishes at 25% confluency (approx. 500K cells). Let attach overnight
?. [Induction of shRNAs]
Add 1000x Doxycyclin to 1 plate of cells with fresh media and induce for 72 hours changing media with fresh doxycyclin daily. Also change media to corresponding non-induced plate
?. [In 96-well plates]
Split cells at density appropriate for cell type. (eg. 5000k cells/well) in total of 150ul of media per well.Make master mixes of cells/well in 50ml conicals and distribute using multi-channel pipette.Add doxyxyclin to induced samples at 500xLet cells attach overnight
?. [In 96-well plates]
Dilute Top2i in media 96-well round bottom plate for transfer to plates with cellsDilution in media without Doxycycline so that both cell samples can get Top2i from the same dilutionsIn our case 2ul of stock concentration of drug (see making stock plate) in 250ul of media (1:125) --> (1:500 when 50uls is added to 150ul already in well. Doxycycline concentration will be diluted in induced samples but was starting with a higher concentration). One row is enough for 1/2 plate. Make multiple rows of dilutions for more plates.
?. [In 96-well plates]
Incubate cells with Top2i for 2 days
?. [In 96-well plates]
Dilute cell titer blue 1:2 in PBSAdd 20ul of cell titer blue to wells and let sit overnight
?. [In 96-well plates]
Transfer suppernatant to 384-well black plates for readingExcite at 544nm and emission of 590nm
TITLE: Isolation of Stromal Vascular Fraction (SVF) from mouse brown adipose tissue (BAT) for single cell RNA-seq
AUTHORS: Farnaz Shamsi, Yu-Hua Tseng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the procedure for the isolation of the Stromal Vascular Fraction (SVF) from mouse brown adipose tissue (BAT) for single cell RNA-seq. This protocol uses a combination of Collagenase I and Dispase II to digest freshly isolated BAT. Compared to using Collagenase I alone, this combination results in a more efficient dissociation of the adipose vasculature.</div></div>

[STEPS]
?. Sacrifice the mouse.
?. Spray the animal extensively with 70 % EtOH and RNaseZap™.
?. Dissect interscapular brown adipose tissue (BAT). If tissues from multiple animals are being dissected, store them in HBSS until all of them are dissected.
?. Mince the tissue to very fine pieces in a 50 ml Falcon tube. Add for each BAT.
[digestion media]
?. Place the tubes in a water bath or incubator with a shaker/rotator at  for .
37 °C
?. Remove the tissue from the incubator and vortex for .
?. Centrifuge at   in a swinging bucket centrifuge.
Centrifuge: 300 34, 10 min, 4 10
?. Aspirate the supernatant carefully not to disturb the pellet of SVF cells.
?. Resuspend the pellets in .
[growth media]
?. Filter through a 100 μm cell strainer into a fresh 50 ml tube. Wash the tube with an additional  and filter through the cell strainer.
10 mL
?. Centrifuge at .
Centrifuge: 300 34, 7 min
?. Completely remove supernatant and re-suspend the pellet in ; place  for .
[sterile ACK lysis buffer]
on ice
?. Filter through a 40 μm cell strainer into a fresh 50 ml tube. Wash the tube with  and filter through the cell strainer.
[growth media]
?. Centrifuge at .
Centrifuge: 300 34, 7 min
?. Resuspend the pellet in .
[%1.5 BSA in PBS]
?. Use  of the cell suspension for cell counting and viability assessment.
10 µl
?. Centrifuge the cell suspension .
Centrifuge: 300 34, 5 min
?. Resuspend the cells in . Incubate the samples for  at .
[dead cell removal bead solution]
37 Room temperature
?. Prepare the binding solution by diluting the 20X solution in sterile ddH2O.
?. Place the MS columns on the MACS separator. Prepare each column by rinsing it with. Let the solution pass through the column.
[1X binding solution]
?. Add  to each sample and apply cell suspension onto the column.
[1X binding solution]
?. Collect effluent in a 2 ml low bind tube as live cell fraction.
?. Rinse the column with an additional .
[1X binding solution]
?. Use  for cell counting and viability assessment.
[sample]
?. Centrifuge the cell suspension .
Centrifuge: 300 34, 5 min
?. Resuspend the cells in  - .
50 µl
[%1.5 BSA in PBS]
?. Keep the cell suspension  and proceed to 10x Genomics Single Cell Protocol. Minimize the time between cell preparation and chip loading.
on ice
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This simple and rapid chromatographic detection method was developed and validated in order to accurately quantify trace levels of free gossypol in different cotton materials, including cottonseed, cottonseed cake and cottonseed cake treated with macrofungi.</p>

[BEFORE_START]
<p>Ice is needed for the sonicate step</p>

[GUIDELINES]
<p>Column and equipment used to develop this method was KINETEX® column and a Waters ACQUITY UPLC H-Class system, respectively. </p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: Colony PCR
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to perform preliminary screening of plasmid-containing colonies without the need for overnight cell culture. This screening aimed to confirm the correct assembly of DNA fragments during Gibson Assembly or the presence of target DNA fragments in the plasmid. After obtaining the results of the colony PCR, the team could then decide whether specific colonies required overnight culturing. This would allow them to perform plasmid extraction the next day, obtain the fully assembled plasmid, and send it out for sequencing to confirm the final structure.

[STEPS]
1. Circle the colony of interest.
 
5. Run a PCR with the setting below:

 

6. After the PCR is complete, proceed to run gel electrophoresis for the samples.
2. Prepare the mixture below in a PCR tube, the final volume is 50 µL:

 

3. Mix the solution well by vortexing the tubes and then split the solution equally into 5 new PCR tubes (each PCR tube would contain 10 µL of the solution).

4. Use an inoculation loop to inoculate some cells from the colony of interest and dunk the inoculation loop into the PCR tube according to the labels. 
7. After gel electrophoresis is finished, put the agarose gel onto a UV Sample Tray, next, put it into the Gel Doc EZ System, and then run the gel imaging program on the desktop.
9. Save the gel image and discard the gel into the biohazard bin.
8. Check if the expected band occurs.
TITLE: JAX - Nuclei Isolation for 10x Genomics A
AUTHORS: William F Flynn, Elise Courtois, Greggory A Perry, Sandra L Daigle, Paul Gabriel, Diane Luo, Jessica Grassmann

[DESCRIPTION]
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the  10x Genomics Multiome assay.  

This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.

This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).

[BEFORE_START]
Familiarize yourself with 10x Genomics protocol CG000365.

[GUIDELINES]
Assess nuclear membrane integrity (refer to 10x Genomics CG000365), using Brightfield or Trypan blue
Assess for debris, if large amount of debris present, filter with smaller pore size to decrease debris. 
Use either Trypan blue and Countess (for clean average size nuclei preps) or AO/PI and Nexcellom K2 counter (if nuclei prep has debris)
If RNA content is of concern Imaging nuclei can be done with fluorescence, and Syto RNA stain


[STEPS]
SECTION: Sample Prep
1. Slightly thaw sample out on ice and place onto pre chilled Petri dish on ice.
SECTION: Sample Prep
2. Cut tissue into small pieces and add to a microcentrifuge tube.
SECTION: Cell lysis
3. In micro centrifuge tube containing the tissue add 100 µL of lysis buffer.

SECTION: Cell lysis
4. Using a plastic pestle to grind tissue by pushing pestle down and twisting to break up the tissue. 
Start timer once lysis buffer is added.
SECTION: Cell lysis
5. Add 25 µL to wash off pestle and place tube on ice for a total of 5 min.

SECTION: Cell lysis
6. After 5 minutes of lysis on ice, 500 x g, 5 min, 4 °C.

SECTION: Cell lysis
7. Remove supernatant and resuspend pellet using wide bore tips in 100 µL.

SECTION: Cell lysis
8. Incubate on ice for a total of 5 min.

SECTION: Washing
9. Add 500 µL and 500 x g, 5 min, 4 °C.

SECTION: Washing
10.   
SECTION: Washing
12. Resuspend sample in 1x Nuclei buffer (10x provided) containing RNase inhibitor, and DTT.
SECTION: Count
9.2. Count using trypan blue or AOPI to get approximate nuclei size and number.
SECTION: Load
13. Load for transposition and continue with the 10x multiome protocol.
SECTION: Washing
9.1.   

SECTION: Washing
11. After last wash, filter suspension through 40um filter.
TITLE: Aqueous adenine solution preparation
AUTHORS: seifip

[DESCRIPTION]
Aqueous adenine solution preparation

[STEPS]
8.    
 


SECTION: Prepare a 10 mM adenine solution in water as follows:
1. Weigh out 0.13513 g of adenine.
SECTION: Prepare a 10 mM adenine solution in water as follows:
2. Transfer the adenine to an Erlenmeyer flask and add an appropriate solvent like distilled water or a biological buffer (amount of solvent will depend on desired final concentration).
SECTION: Prepare a 10 mM adenine solution in water as follows:
3. Stir the solution using a magnetic stirrer to help dissolve the adenine. If the adenine is not fully dissolving at room temperature, you can heat the solution gently in a water bath while stirring to enhance dissolution.
SECTION: Prepare a 10 mM adenine solution in water as follows:
4. If there remains undissolved adenine, transfer your solution into evenly divided and balanced centrifuge tubes. Centrifuge at moderate speed (for example, 3000 to 5000 rpm) for a short period of time (around 5 to 10 minutes).
SECTION: Prepare a 10 mM adenine solution in water as follows:
5. After centrifugation, a pellet of undissolved adenine may have formed at the bottom of the tube. The clearer liquid on top, or the supernatant, is your solution of dissolved adenine. Carefully pipette off the supernatant to a new container, avoiding the pellet.
TITLE: TEM
AUTHORS: Alexander Röntgen

[DESCRIPTION]
TEM protocol.

[STEPS]
SECTION: Preparation
1. Recover samples by pipetting out of aggregation plate
SECTION: Preparation
2. Glow-discharge grids (3-mm 300-mesh (EM Resolutions Ltd))
SECTION: Sample spotting and staining
3. Take up grid with tweezers
SECTION: Sample spotting and staining
4. Spot 2.3 µL sample on grid
SECTION: Sample spotting and staining
5. Stain sample with 2.3 µL 2% uranyl acetate for 40 s at room temperature
SECTION: Imaging
6. Image grids on an electron microscope (FEI Tecnai G2 transmission electron microscope)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p align="LEFT"><span style="font-family: Arial-BoldMT; font-size: small;">Lipofectamine</span><span style="font-family: Arial-BoldMT; font-size: xx-small;">™ </span><span style="font-family: Arial-BoldMT; font-size: small;">2000 Transfection Protocol</span></p>
<p align="LEFT"> </p>
<p align="LEFT">Description</p>
<p> </p>
<p align="LEFT"><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">Lipofectamine</span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">™ </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">2000 is a proprietary formulation for the transfection of nucleic</span></p>
<p align="LEFT">acids (DNA and RNA) into eukaryotic cells providing the following advantages:</p>
<p align="LEFT"><span style="font-family: SymbolMT; font-size: xx-small;">• </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">Highest transfection efficiency in many cell types and formats (</span><em><span style="font-family: PalatinoLTStd-Italic; font-size: xx-small;">e.g. </span></em><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">96-well).</span></p>
<p align="LEFT"><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">Refer to the Cell Lines database at </span><span style="color: #800080; font-family: PalatinoLTStd-Roman; font-size: xx-small;">www.invitrogen.com </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">for a list of cell types</span></p>
<p align="LEFT">successfully transfected.</p>
<p align="LEFT"><span style="font-family: SymbolMT; font-size: xx-small;">• </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">Nucleic acid-Lipofectamine</span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">™ </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">2000 complexes can be added directly to cells in</span></p>
<p align="LEFT">culture medium, in the presence or absence of serum.</p>
<p align="LEFT"><span style="font-family: SymbolMT; font-size: xx-small;">• </span><span style="font-family: PalatinoLTStd-Roman; font-size: xx-small;">It is not necessary to remove complexes or change/add medium after</span></p>
<p>transfection, but complexes may be removed after 4-6 hours.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/DNase-I-Treatment-c3myk5" target="_blank">DNase I Treatment</a> protocol for DNase inactivation.

[GUIDELINES]
Note: EDTA tetrasodium salt dihydrate is somewhat more soluble and can be made more easily to 1.5M. FW is slightly more so weigh out 3.80 g and follow directions above.

[STEPS]
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TITLE: Minimal important difference in SD units
AUTHORS: Yasushi  Tsujimoto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The current study aims first to describe the anchor-based minimal important difference (MID) estimates in standard deviation (SD) units and then to examine if the robustness of such estimates depends on the SD to be used or on the methodological quality of the anchor-based estimates.</div></div>

[STEPS]
TITLE: Plasmid Extraction (Plasmid Isolation)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to isolate the plasmid from the cells.

[BEFORE_START]
Glycerol stock (900 µL of cell stock in 300 µL of 100% glycerol solution) may be prepared and keep it in the -80 °C fridge before the plasmid extraction procedure.


[STEPS]
SECTION: Cell Culture
1. Incubate the cells containing the plasmid of interest in a Falcon tube with 5 mL of LB media and 5 µL of the appropriate antibiotics   at 37 °C before starting the plasmid extraction.

SECTION: Plasmid Extraction
2. Take out the Falcon tube with the cultured cells from the incubator.
SECTION: Plasmid Extraction
3. Centrifuge the Falcon tube at 5000 rpm, 5 min, 4 °C .

SECTION: Plasmid Extraction
4. Discard the supernatant in the Falcon tube and keep the cell pellet.
SECTION: Plasmid Extraction
5. Add 250 µL of   (with  added, kept in the 4 °C fridge) into the Falcon tube and resuspend the cell pellet.

SECTION: Plasmid Extraction
6. Transfer the whole solution into a Eppendorf tube.
SECTION: Plasmid Extraction
7. Add 250 µL of    into the Eppendorf tube to lyse the cells.

SECTION: Plasmid Extraction
8. Shake the Eppendorf tube for mixing.
SECTION: Plasmid Extraction
9. Add 350μL of   into the Eppendorf tube.
SECTION: Plasmid Extraction
10. Shake the Eppendorf tube for mixing, a cloudy solution shall be observed.
SECTION: Plasmid Extraction
11. Centrifuge the Eppendorf tube at 13 rpm, 10 min.

SECTION: Plasmid Extraction
12. Transfer the supernatant into a Mini Prep tube and discard the cell debris.
SECTION: Plasmid Extraction
13. Centrifuge it at 13 rpm, 1 min.

SECTION: Plasmid Extraction
14. Discard the flow-through and place the Mini Prep tube back into the same tube.
SECTION: Plasmid Extraction
15. Add 500 µL of   into the Mini Prep tube.

SECTION: Plasmid Extraction
17. Discard the flow-through and place the Mini Prep tube back into the same tube.
SECTION: Plasmid Extraction
18. Add 700 µL of   into the Mini Prep tube.

SECTION: Plasmid Extraction
20. Discard the flow-through and place the Mini Prep tube back into the same tube.
SECTION: Plasmid Extraction
22. Transfer the Mini Prep column into the newly labelled Eppendorf tube.
SECTION: Plasmid Extraction
23. Add 50 µL of DI water into the Eppendorf tube.

SECTION: Plasmid Extraction
25. Discard the Mini Prep column, the solution left in the Eppendorf tube is the plasmid.
SECTION: Plasmid Extraction
26. Use NanoDrop
 
to measure the concentration (under "dsDNA" mode) and the purity of the plasmid. 
SECTION: Plasmid Extraction
27. Store the isolated plasmid at room temperature.
SECTION: Plasmid Extraction
16. Centrifuge it at 13 rpm, 1 min.

SECTION: Plasmid Extraction
19. Centrifuge it at 13 rpm, 1 min.

SECTION: Plasmid Extraction
21. Centrifuge it at 13 rpm, 1 min to remove the   residual.

SECTION: Plasmid Extraction
24. Centrifuge it at 13 rpm, 1 min, ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.
TITLE: VU TIS Multimodal Molecular Imaging Pipeline for KPMP Biopsy Interrogation 
AUTHORS: Katerina V Djambazova, Lukasz Migas, Martin Dufresne, Jamie Allen, N. Heath Patterson, Léonore Tideman, Ellie Pingry, Madeline E. Colley, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins

[DESCRIPTION]
This protocol provides an overview of the Multimodal Molecular Imaging pipeline used by the Vanderbilt University Tissue Interrogation Site (VU TIS) as part of the Kidney Precision Medicine Project (KPMP). Individual protocols are contextualized within our larger workflow.

[STEPS]
SECTION: Sample Interrogation Assays
2. Autofluorescence Microscopy (AF) is collected on all tissue sections prior to IMS
AF QC Protocol: AFQC Protocol
AF protocol: AF Microscopy
 

SECTION: Sample Interrogation Assays
3. MALDI Imaging Mass Spectrometry (IMS)
Matrix Deposition Protocol (Sublimation): Tissue Washing and Matrix deposition Protocol
High Spatial Resolution MALDI IMS Data Acquisition: MALDI IMS Protocol
SECTION: Sample Interrogation Assays
4. LC-MS/MS Lipidomics 
LC-MS/MS is performed on a single serial biopsy section.
Protocol: LC-MS/MS Protocol
SECTION: Sample Interrogation Assays
5. Post-IMS Autofluorescence Microscopy 
Protocol: Post-IMS AF
SECTION: Sample Interrogation Assays
6. PAS Staining 
Matrix removal and Staining: PAS Staining Protocol
SECTION: Data Pre-Processing and Analysis
7. Image registration - AF, PAS and IMS 
Protocol: Image Registration
 

SECTION: Tissue Screening and Assessment
1. Asses Kidney Tissue Sections

 Sample Processing Go/No-Go Criteria 
     Sample Processing   Go/No-Go Criteria      Reasoning        Biopsy must be LN2 core      Other storage conditions can induce   analyte suppression effect in MALDI IMS analysis        Biopsy size must be ³ 3mm      Smaller tissue sections are difficult to   handle, section, and mount        Time to preservation must be < 10 min      Prolonged exposure prior to preservation   can cause metabolite/lipid degradation   
 On-Site Tissue Integrity Considerations
Tissue QCReasoning / Data FlagsAre there any glomeruli in the biopsy section?Ensures proper coverage of biological featuresVisible cracks/folds on the tissue surfaceThis can lower FTU segmentation performance, due to non-segmentable featuresVisible freezing artifacts on the tissue surfaceFreezing artifacts can induce analyte delocalization, jeopardizing MALDI IMS spatial integrity

 
SECTION: Data Pre-Processing and Analysis
10. Analyte Annotation
Tentative lipid annotation from the MALDI IMS data
Protocol: Tentative Lipid Annotation
SECTION: Data Pre-Processing and Analysis
9. Functional Tissue Unit (FTU) Segmentation
Protocol: FTU Segmentation
 

SECTION: Data Pre-Processing and Analysis
8. Data Pre-Processing
Protocol: MALDI IMS Data Pre-Processing