eamag/protocols-with-steps huggingface dataset:
TITLE: Basketball Exercise
AUTHORS: Ziyong Ma

[DESCRIPTION]
A simple basketball training plan.

[GUIDELINES]
Just try your best to finish it

[STEPS]
SECTION: Dribble Exercise
2. Ball Rubs
1 x 10 s 

SECTION: Dribble Exercise
3. Single Leg Wraps
-Left 1 x 20 s 
-Right 1 x 20 s 

SECTION: Dribble Exercise
4. 3-3-3 Dribble Drill
 2 x 20 s 
PS: with variations
SECTION: Dribble Exercise
5. Cone Dribbling
1 x 3 min 
-with variations

SECTION: Shooting Exercises
6. Form Shooting (one hand)
-Left 1 x 20 s 
-Right 1 x 20 s 
SECTION: Warm Up
1. Run To That Lines
3 min 

SECTION: Shooting Exercises
7. 1-2 Steps Lay-Ups
1 x 2 min for each side

SECTION: Shooting Exercises
8. Shooting Line Game (Games to 8 makes)
SECTION: Cometing
9. Dribble Relays
2 x 

SECTION: Cometing
10. 3 on 3 Games
(games to 5)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>On June 14-17, 2018, the 23rd EHA conference was held in Sweden-Stockholm. The heavy research in the blood field took place. The development of new drugs in the field of acute leukemia has progressed rapidly. Among them, antibody-drug conjugates(<a href="https://www.creative-biolabs.com/adc/services.htm" target="_blank" rel="noopener noreferrer"><u>ADC</u></a>) including Gemtuzumab Ozogamicin targeting CD33 and Inotuzumab targeting CD22 showed significant effects in adult acute myeloid leukemia and adult acute lymphoblastic leukemia, respectively.</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The procedures to determine the half-lives of proteins by pulse-chase analysis and cycloheximide blocking are well established. In the case of oligomeric proteins, these methods do not differentiate whether the stabilities of the monomeric versus oligomeric forms of the protein differ. This method describes how to determine the half-lives of oligomeric proteins that are stable in the presence of low concentrations of the detergent SDS by combining cycloheximide blocking with non-dissociating Western blot (Dutta et al., 2011). The addition of cycloheximide inhibits protein synthesis and non-dissociating Western blot uses low concentrations of SDS to separate stable oligomers from monomers. The amount of the protein of interest detected in samples taken at different times after adding cycloheximide can be used to determine the half-life of the oligomeric protein.</p>
<p> </p>
<p>References</p>
<ol>
<li>Byk, L., Iglesias, N., De Maio, F., Gebhard, L., Rossi, M. and Gamarnik, A. (2016). Dengue virus genome uncoating requires ubiquitination. <em>mBio</em>, 7(3), pp.e00804-16.</li>
<li>Dutta, D., Chattopadhyay, S., Bagchi, P., Halder, U., Nandi, S., Mukherjee, A., Kobayashi, N., Taniguchi, K. and Chawla-Sarkar, M. (2011). Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3). <em>Journal of Biological Chemistry</em>, 286(22), pp. 20065-20077.</li>
<li>Elroy-Stein, O., Moss, B., 2001. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. <em>In: Current Protocols in Molecular Biology, </em>chapter 16, unit16 9.</li>
<li>Ward, G.A., Stover, C.K., Moss, B., Fuerst, T.R., 1995. Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proceding of the Nationall Academy of Sciences U S A, 92, pp. 6773-6777.</li>
</ol>

[STEPS]
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TITLE: Plasmid Extraction
AUTHORS: Hung Liang Pai, Huan Jui Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for extracting plasmid from E. coli DH5-Alpha strains. </div></div>

[STEPS]
?. [Preparation]
Set the dry bath incubator at
37 °C
?. [Preparation]
Prepare Presto™ Mini Plasmid Kit
?. [Preparation]
Take out the centrifuge tube containing medium with bacteria that has been cultured overnight.
?. [Protocol]
Centrifuge the centrifuge tube under this condition -   .
Centrifuge: 15000 34, 6 min
?. [Protocol]
Discard the supernatent
Recommendation: Keep the bacterial pellet away from supernatent if plausible to insure minimum loss of bacteria.
?. [Protocol]
Add  of PD1 reagent to the centrifuge tube. Vortex the tube to completely mix bacterial pellet with PD1.
200 µl
?. [Protocol]
Add   of PD2 reagent to the centrifuge tube. Shake the tube gently and let it stand for  to .
200 µl
Warning: Do not vortex the mixture after adding PD2!!!!
?. [Protocol]
Add   of PD3 reagent to the centrifuge tube. Shake the tube gently and centrifuge the tube under this condition -  .
300 µl
Centrifuge: 16000 34, 10 min
Warning: Do not vortex the mixture after adding PD3!!!!Tips: Prepare the PDH column when centrifuging.
?. [Protocol]
After centrifugation, transfer the supernatent into a PDH column and discard the pellet. Centrifuge the PDH column under  and discard the flowthrough liquid in the collection tube.
Centrifuge: 15000 34, 30 s
?. [Protocol]
Add  of Wash 1 Buffer into the PDH column and centrifuge under . Discard the flowthrough in the collection tube.
400 µl
Centrifuge: 15000 34, 30 s
?. [Protocol]
Add   of Wash 2 Buffer into the PDH column and centrifuge under  .Discard the flowthrough in the collection tube.
600 µl
Centrifuge: 15000 34, 30 s
?. [Protocol]
Add   of Elution Buffer into the middle of PDH column and incubate the PDH column in a drybath incubater under  .
50 µl
37 °C
Recommendation: Add the Elution Buffer directly onto the silicon membrane to insure optimal euting efficacy.
?. [Protocol]
Centrifuge again under   and replace the collection tube with a new eppendorf. (Discard the flowthrough)
Centrifuge: 15000 34, 3 min
?. [Protocol]
Centrifuge under . The flowthrough in the eppendorf contains plasmids derived from the cultured bacteria.
Centrifuge: 15000 34, 2 min
Recommedation: Proceed to measure the concentration of plasmid.
TITLE: Protocol 2: PCR Wet Lab 
AUTHORS:  

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

[STEPS]
?. DNA ExtractionDNA Extraction: Saliva (Bento lab)  This protocol extracts DNA from Saliva using a microcentrifuge and thermocycler. The microcentrifuge separates the cells in the sample into a pellet at the bottom of the microcentrifuge tube. Extra liquid is drawn out of the microcentrifuge tube. Cells are then heated in the thermocycler and placed in the microcentrifuge to separate DNA from other cellular material.
?. PCRLactose intolerance: PCR (Bento lab)Bitterness tasting: PCR (Bento lab)Athlete gene: PCR (Bento lab)This protocol uses a thermocycler in order to amplify sections of DNA genes. Students will amplify genes specific to lactose intolerance, bitterness tasting, and an athletic gene. DNA template is extracted from the previous protocol and added to a PCR master mix and designated primer. PCR master mix is composed of Taq polymerase, dNTPs, MgCl2, and pH buffer. After adding all reagents, students will set parameters for the thermocyclers. Each PCR experiment will have different parameters due to the nature of its respective primers.
?. Gel ElectrophoresisGel electrophoresis (Bento lab)This protocol uses a gel electrophoresis to identify the amplified sequences from the previous protocol, PCR. Students will set up their gel electrophoresis by creating an agarose gel with necessary lanes, adding 0.5X TBE buffer, and attaching electrodes. After set up, DNA ladders and amplified DNA will be added to lanes in the gel. The gel will run at 50V for 40 minutes. Then students will analyze the results and determine their allele type and phenotype.
?. Take a picture of your gel electrophoresis results and upload it to your Lab Notebook.
TITLE: Initial Rapid Pathology Assessment of Kidney Tissue
AUTHORS: Jamie Allen, Carrie  Romer, Elizabeth Neumann, Maya Brewer, Haichun Yang, Jeff Spraggins, Danielle Gutierrez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">Prepare formalin fixed tissue with freezing fresh tissue protocol (dx.doi.org/10.17504/protocols.io.6wghfbw).  </div><div class = "text-block">This protocol provides the steps to prepare formalin fixed human kidney tissue and perform histology assessment for normalcy.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Pathological assessment of kidney tissue for use in LC-MS/MS and imaging.</div></div>

[STEPS]
?. Tissue is embedded in protocol: dx.doi.org/10.17504/protocols.io.br4fm8tn
?. Section samples at 5 µm on a microtome.
?. PAS stain tissue sections with protocol: dx.doi.org/10.17504/protocols.io.buaknscw
?. Scan slides with brightfield scanner (Leica) and save as .tiff or .jpg
?. Place saved images on QuPath for analysis.
?. Assess and record the following information for each tissue:(%): cortex and medulla(Yes/No): Pyramid Presence, Autolysis, Non-Renal disease (i.e. cancer)(0=None – 3=Severe): Glomerular disease, Tubulointerstitial disease
?. Based on FFPE assessment, kidneys will be used accordingly:LC-MS/MS normalcy: low or no glomerular disease, and 75% or more cortex for normal100% tumor for diseased3D Imaging: 50:50 cortex:medulla low or no glomerular diseaseno renal disease
TITLE: PCR amplification of long GC-rich DNA targets
AUTHORS: Nadia Assal, Min Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol allows the amplification of long and high GC content genes by PCR without the need for many optimizations. In this protocol, the</span><span style = "font-style:italic;"> Mycobacterium bovis</span><span>, a genome with more than 60% GC content, is used as a model and a source of source for a long gene (>1.5 kb) and a GC content of 77.5%. The protocol works well irrespective of the length of the primers.   </span></div></div>

[STEPS]
?. Extract M. bovis genomic DNA using according to the manufacturer’s instructions for Gram-positive bacteria. Use 5 ng/µl of genomic DNA was used in each reaction
?. Prepare the master mix for the polymerase enzyme as follows:  AB1Componentvolume in µl2Milliq Water29.53Buffer104DNTPs&nbsp; (2.5mM)45DMSO2.56Primers Mix
  (Forward +Reverse) 25 µM27Genomic
  DNA(5ng/ul)18Polymerase19Total&nbsp;50 Prepare your mix on ice. Always vortex and centrifuge all reagents before use. Add the enzyme last. Vortex and mix your reaction  tubes. Make sure there are no bubbles.
AB1Componentvolume in µl2Milliq Water29.53Buffer104DNTPs&nbsp; (2.5mM)45DMSO2.56Primers Mix
  (Forward +Reverse) 25 µM27Genomic
  DNA(5ng/ul)18Polymerase19Total&nbsp;50
?. Adjust the thermal cycler as follows:Use  a 2 Step Protocol:35-40 cycles of (Denaturation at 98 °C for 15 seconds then Annealing and extension at 68 °C for 1min / kb)the reaction is then kept on hold at 4 °C Adjust the speed of the thermal cycler heating and cooling ramp speed to 2 °C/seconds.Load the thermal cycler with your tubes. Start your program
?. After finishing your program, run your reactions on agarose gel electrophoresis.
TITLE: Marchantia spores sterilisation
AUTHORS: Linda Silvestri, Eftychis Frangedakis, Marius Rebmann, Susana Sauret-Gueto

[STEPS]
?. Add one Milton tablet into 25 mL of sterile water to prepare the "sterilization solution".
?. Add the sporangia into a 1.5 mL centrifuge tube and add 0.5 mL of sterilization solution into the tube (B in Figure)
?. Use sterile metal tweezers to crush the sporangia (C in Figure).
?. Place a 40 μM cell strainer into a 50 mL Falcon tube and pour the 0.5 mL suspension of crushed spore heads onto the filter and then wash the strainer with another 1 mL of sterilization solution (D in Figure).
?. Once filtered, aliquot the spore solution in 1.5 mL Eppendorf tubes (1.5 mL per tube) and incubate at room temperature for 10 min (the exposure to the sterilization solution should not exceed 25 min) (E in Figure).
?. Centrifuge for 2 min at 15800 xg at room temperature, discard the supernatant and re-suspend the spores in sterilization solution (150 μl per transformation planned) (F in Figure).
?. Spread 150 μl of re-suspended spores on a plate containing growth media (G in Figure), seal the lid with micropore tape and place under continuous light at 21 °C 150 μmol/m2/s for 7 days (H in Figure). Incubate with the bottom side up, which will prevent rhizoids from growing into the agar, making the removal of sporelings from the plate easier by simply using a sterile scalpel.
?.
TITLE: Cartilage staining
AUTHORS: Satheeswaran Balasubramanian, Ekambaram Perumal

[DESCRIPTION]
The Alcian Blue staining technique is widely used among developmental biologists to observe the embryonic development of cartilage and bone structures in embryos and complete zebrafish larvae. Alcian blue is a positively charged dye that is thought to stain cartilage through an electrostatic interaction with negatively charged acidic mucopolysaccharides (Scott et al. 1996). The principle of this method is that different concentrations of magnesium ions can be used to compete with alcian blue for the negative charges of acidic mucopolysaccharides to differentiate different types of muco substances.

[BEFORE_START]
Every protocol needs to be validated in the laboratory when first introduced. The present protocol describes validation steps that were taken in the Molecular Toxicology lab, at Bharathiar University. 
Always wear personal protective equipment. 
All the steps are optimized for 24 well plate for multiple groups such as control and treated. Each solution can be added to the well plate for convenience.

[GUIDELINES]
This protocol is optimized for 4 days post-fertilization of zebrafish larvae. It can be adapted for higher dpf.

[STEPS]
SECTION: Larval fixation
1. At the desired stage, the larvae for staining are taken and washed once in PBS for 5 minutes.
SECTION: Larval fixation
2. The larvae were euthanized using the cold shock method (Keep at 4oC for 5-10 minutes).
SECTION: Larval fixation
3. Transfer the euthanized larvae into 4% PFA and keep it in the rocker for 2 hours at room temperature (if required, it can be kept overnight at 4oC).
SECTION: Larval fixation
4. After fixation, remove the 4% PFA and wash with PBST for 5 minutes, two times.
SECTION: Larval fixation
5. Dehydrate using 50% ethanol and keep it in the rocker for 10 minutes at room temperature.
SECTION: Staining
6. After dehydration, add 1 ml of alcian blue staining solution and keep it in a rocker overnight at room temperature.
SECTION: Bleaching
7. Remove the stain solution and rinse once using PBST.
SECTION: Bleaching
8. Transfer the larvae to a bleach solution and incubate in dark at room temperature. Assess the bleaching process (using a microscope) and stop the reaction when the larvae become transparent (approximately min for 24 hpf or 20-30 min for 96 hpf and above).
SECTION: Clearing
9. Remove the bleach solution and rinse once using PBST.
SECTION: Clearing
10. Clear the beached larvae with 25% glycerol and 0.25% KOH. Keep them in a rocker at room temperature for 30 minutes to overnight.
SECTION: Clearing
11. Replace the solution with 50% glycerol and 0.25% KOH. Keep them in a rocker at room temperature for 2 hours to overnight.
SECTION: Storage
12. Store larvae in a solution of 50% glycerol and 0.1% KOH at 4 oC. Avoid long-term storage as it will diminish the stain. Capture images of the cartilage using any bright field microscope
SECTION: Results
13.
TITLE: NAN KB Demo: Data download
AUTHORS: Abby   Moore

[DESCRIPTION]
The purpose of this protocol is to describe how data may be downloaded from "our project."

The scope of this protocol extends to any instance where a user must download data from a spectrometer that is a part of "our project."

[BEFORE_START]
For trained users, this protocol requires an NMRBox account as well as a virtual machine program.

[GUIDELINES]
This protocol may be performed by "our project" staff, trained users, and non-trained collaborators who wish to download their data.

[STEPS]
SECTION: Data download
1. Select the appropriate case below to proceed.
SECTION: Data download
2. Open a terminal in 
 

SECTION: Data download
3. Copy your data from the spectrometer using the following command: 
 

SECTION: Data download
4. You will be asked for your account password - type this in and hit enter, your download should commence immediately.


 

SECTION: Data download
2. An NMR staff member who ran your experiments can send you the data from the spectrometer for processing – please reach out to the appropriate parties.
TITLE: Patient PBMC flow cytometry
AUTHORS: rebeccawallings

[DESCRIPTION]
Patient PBMC flow cytometry

[STEPS]
1. 1 x 106 PBMCs were taken for flow cytometry and transferred to a v-bottom 96-well plate
(Sigma, CLS3896-48EA) and centrifuged at 300x g for 5 minutes at 4°C.

Cells were resuspended in 50 µL of PBS containing diluted fluorophore-conjugated antibodies and incubated in the dark at 4°C for 20 minutes. Cells were centrifuged at 300x g for 5 minutes at
4°C and washed in PBS x 2. Cells were fixed in 50 µL of 1% paraformaldehyde (PFA) at 4°C in the dark for 30 minutes. 

Cells were centrifuged at 300x g for 5 minutes and resuspended in 200 µL FACs buffer (PBS, 0.5 mM EDTA, 0.1% sodium azide). 

Cells were taken for flow cytometry on a MACS Quant Analyzer (Miltenyi). A minimum of 100,000 events were captured per sample and data were analyzed using FlowJo version 10.6.2 software (BD Biosciences). 

When validating flow cytometry panels and antibodies, fluorescence minus one controls (FMOCs) were used to set gates and isotype controls were used to ensure antibody-specific binding.
TITLE: SynBot Protocols
AUTHORS: Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu

[DESCRIPTION]
Protocols related to the paper SynBot: An open-source image analysis software for automated quantification of synapses. Includes details of rat neuron and astrocyte culture, in vitro synapse staining, in vivo synapse staining, and using SynBot with manual or ilastik thresholding.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The purinosome has been observed in a broad spectrum of cells, but some studies claim that it is an artefact of the constructs used for visualization or stress granules resulting from the exposure of cells to nutrient-reduced growth media. Both may be true depending on the method of observation. To clarify this point, we combined two previously used methods, transfection and immunofluorescence, to detect purinosomes in purinosome-free cells deficient in particular DNPS steps (CR-DNPS cells) and in cells deficient in the salvage pathway, which resulted in construction of the purinosome regardless of purine level (CR-HGPRT cells).</p>
<p>To restore or disrupt purinosome formation, we transiently transfected CR-DNPS and CR-HGPRT cells with vectors encoding BFP-labelled wild-type (wt) proteins and observed the normalization of purinosome formation. The CR-DNPS cell line transfected with a DNA plasmid encoding an enzyme with zero activity served as a negative control for purinosome formation. </p>

[STEPS]
?.
TITLE: MBP-Clu-tail purification from Escherichia coli cells
AUTHORS: Andreas Bracher, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently purify the fusion protein Maltose binding protein (MBP)-Clu-tail (204-238) from Escherichia coli.

[STEPS]
SECTION: His6-Ubiquitin-GFP-Clu-tail expression and cell lysis
1. Express MBP-Clu-tail in E. coli Bl21 (DE3) codon+RIL cells cultured in 1 L LB Medium containing 2 1655 glucose at 37 °C with 1 millimolar (mM) IPTG during 120 min.

SECTION: His6-Ubiquitin-GFP-Clu-tail expression and cell lysis
2. Centrifuge culture and keep pellet.
SECTION: His6-Ubiquitin-GFP-Clu-tail expression and cell lysis
3. Re-suspend pellet in 25 mL volume of ice-chilled binding buffer, add Complete protease inhibitor cocktail (Roche) and 1 millimolar (mM) phenylmethylsulfonyl fluoride (PMSF).

SECTION: Amylose affinity chromatography
6. Load supernatant onto a 20 mL Amylose Resin (New England Bioloabs) column previously equilibrated with binding buffer by gravity flow at 4 °C.

SECTION: Amylose affinity chromatography
7. Wash the column with 12 CV of ice-chilled binding buffer.
SECTION: Amylose affinity chromatography
8. Elute MBP-Clu-tail protein with 12x 3 mL of ice-chilled Elution buffer. Collect fractions of 3 mL volume. Store fractions on ice.

SECTION: Amylose affinity chromatography
9. Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
 

SECTION: Amylose affinity chromatography
10. Pool fractions containing MBP-Clu-tail.
SECTION: Amylose affinity chromatography
11. Concentrate pool to less than 10 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C.

SECTION: Size exclusion chromatography
12. Apply concentrate on a HiLoad 26/600 Superdex-200 (Cytiva 28-9893-36) column equilibrated with PBS. Develop the column at 4 °C and collect 10 mL fractions.

SECTION: Size exclusion chromatography
13. Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
 

SECTION: Size exclusion chromatography
14. Merge fractions with MBP-Clu-tail peak. Concentrate to 1.5 mL volume by ultrafiltration using 10 kDa cut-off spin concentrator at 4 °C, aliquot and flash-freeze purified MBP-Clu-tail in liquid nitrogen for storage at -70 °C.
 

 

 



SECTION: His6-Ubiquitin-GFP-Clu-tail expression and cell lysis
4. Lyse cells by ultrasonication in ice bath (10 cycles of 30 s ultrasonication with 90 s intermittent cooling).
SECTION: His6-Ubiquitin-GFP-Clu-tail expression and cell lysis
5. Clear lysate by centrifugation at 22000 rpm in a JA25.50 rotor at 4 °C.
TITLE: Coating Slides with Gelatin
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes how 1x3 glass microscope slides are coated with gelatin to improve adherence of fixed adult mouse brain sections or other tissue to slides.</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: No Title
AUTHORS: 

[DESCRIPTION]
This is a very simple protocol&nbsp;showing how to extract haemolymph from adult Drosophila melanogaster. (Based on protocols from Sigma Aldrich).

[GUIDELINES]
<span class="s1"><strong>Materials:</strong></span> <br /><span class="s1">-0.5 &mu;l capillaries with adequate suction cup<br /></span> <span class="s1">-0.5ml and 1.5ml Eppendorf cap</span> <br /><span class="s1">-Ice</span> <br /><span class="s1">-Peaked stylus</span> <br /><span class="s1">-2 forceps (or scissors) to clip wings</span>

[STEPS]
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