eamag/protocols-with-steps huggingface dataset:
TITLE: Goga Lab RT-qPCR protocol: QuantStudio6 
AUTHORS: Jeremy.williams

[DESCRIPTION]
Guidelines for preparing RT-qPCR samples for QuantStudio 6 located in HSW7 lab space.

[BEFORE_START]
*Thaw on ice (leave time!) and keep all reagents on ice through preparation. Prepare the plate on ice.


[GUIDELINES]
*start with full tip boxes and use tips in coordination with your plate map, so you never get lost
*watch 1uL volumes in the pipet tip like a hawk - major source of variability
*cap and gently vortex your *mixture per primer set* every 12 replicates or so, sometimes things separate

[STEPS]
1.   Dilute stock IDT primers to 100uM

2. Mix your forward and reverse primer pairs together, to a final dilution of 10uM forward and 10uM reverse. For example, add 10uL each of forward and reverse primers to 80uL PCR-quality DI for 100uL final volume.
3. Mix *total reagent volumes required* (see excel spreadsheet, green) for DI and PowerUP
4. Mix *mixtures per primer set* (blue) volumes together.
5. Pipet 1uL diluted cDNA into respective qPCR well, aiming for the sidewall of each respective well.
6. Add 19uL *mixture per primer set* into each respective well.
7. Seal plate, and spin down 1000rpm for 1 minute. Use bacterial, not tissue culture, centrifuge.   
8. Load plate into centrifuge and proceed using QuantStudio software suite.
TITLE: SOLUTION- 09 - Trypan Blue solution
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recepe is used in the following protocols:</div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01a - </span><span>Isolation of Human PBMC from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01b - </span><span>Isolation of Human PBMC from Whole Blood</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 02 - </span><span>CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 03 -</span><span> TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01a - </span><span>Isolation of Human PMN from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01b - </span><span>Isolation of Human PMN from Whole Blood</span></div></div>

[STEPS]
TITLE: Insect Vision 1.0.1
AUTHORS: Evripidis Gkanias, Benjamin Risse, Michael Mangan, Barbara Webb

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h2>InsectVision</h2></div><div class = "text-block">Computer Vision Toolbox for Insect Vision</div><div class = "text-block"><h2>Version 1.0.1</h2></div><div class = "text-block">This version has been created as support for the article:</div><div class = "text-block">Gkanias E., Risse B., Mangan M., and Webb B. (2019) From skylight input to behavioural output: a computational model of the insect polarised light compass. PLOS Computational Biology.</div><div class = "text-block"><span>To be able to run all the experiments and replicate the results presented in the article, the </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">compmodels</span></a><span> package is needed. If you don't have access to this package, please contact the authors.</span></div><div class = "text-block"><span>Clone both repositories and set the </span><span style = "font-weight:bold;">compmodels</span><span> as a dependence to the </span><span style = "font-weight:bold;">insectvision</span><span> package.</span></div><div class = "text-block"><span>To see the results, it is not necessary to run the code. By simply opening the </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">notebooks/plos.ipynb</span></a><span> file, the plots should be automatically generated for you to observe them.</span></div><div class = "text-block"><span>To create the plots by yourself, you need to start a </span><span style = "font-weight:bold;">Jupyter notebook</span><span> kernel at the root of the package. Then run the </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">notebooks/plos.ipynb</span></a><span> file, which already contains all the plots and the respective code to replicate them. Some plots (especially the ones related to the global optimisation) may need a long time to run; this does not mean that they do not work.</span></div><div class = "text-block"><span>All the code has been implemented by </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Evripidis Gkanias</span></a><span style = ":;">.</span></div><div class = "text-block"><span>You can go the github repository by following </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">this</span></a><span> link. The release version is attached and it can also be found </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div><div class = "text-block"><span>Copyright © 2019 Gkanias el al. This is an open access article distributed under the terms of the </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Creative Commons Attribution License</span></a><span>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</span></div></div>

[STEPS]
TITLE: SPOT2 protocol
AUTHORS: Huimin Zhao, stlane2 , Guanhua Xun

[STEPS]
?. Label test kit bag with patient's name and a unique sample number for the test run.  Provide a 20 uL microcap and PCR tube and have patient collect a saliva sample and place it into then seal the PCR tube.  Label PCR tube with sample number matching the test kit bag.
?. Place PCR tubes into floating rack and insert into sous vide water bath at 95C.  Incubate for 5 minutes for sample pretreatment.
95 °C
?. During incubation, scan barcodes on test kits and enter patient identifying information into the SPOT2 device.
?. After completion of pretreatment, use the second provided 20 uL microcap to transfer a small volume of saliva from the PCR tube to the capillary tube, containing SPOT assay mastermix.  Place capillary tubes into floating rack and insert into sous vide water bath at 63C for 30 minutes.SPOT assay mastermix:  ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
63 °C
ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
2 µl
1 µl
8 µl
5.6 µl
3.2 µl
0.8 µl
?. Transfer floating capillary rack to sous vide at 95C for 5 minutes.
95 °C
?. Individually transfer capillary tubes to SPOT2 device and press "Run" on the SPOT software.  An integrated RFID chip on each capillary will automatically link test result to the patient information entered previously.
TITLE: Isolation of PBMCs From Whole Blood
AUTHORS: Gerardo Ongari

[DESCRIPTION]
This protocol details methods for the isolation of peripheral blood mononuclear cells (PBMCs) from Whole Blood

[BEFORE_START]
Pre-procedure: Ensure all reagents are at room temperature (15 - 25°C). The procedure must be carried out under a cell culture hood. 

[STEPS]
SECTION: Step 1: Plasma Isolation
1. Collect 36 mL whole blood into EDTA tubes

SECTION: Step 1: Plasma Isolation
2. Centrifuge the blood samples at 1000 x g, 20 min, 25 Room temperature 

SECTION: Step 1: Plasma Isolation
3. Carefully remove collection tubes from centrifuge; plasma is the top layer above the rest of the whole blood layer
SECTION: Step 1: Plasma Isolation
4. Using caution not bring up the remaining whole blood layer, remove plasma and aliquot it into 1.5 mL microcentrifuge tubes

SECTION: Step 1: Plasma Isolation
5. Centrifuge the isolated plasma at 1600 x g, 20 min, to remove residual cells and debris

SECTION: Step 1: Plasma Isolation
6. After centrifugation, aliquot the plasma supernatant into 1mL aliquots  by using microcentrifuge tubes labeled with patient ID
SECTION: Step 1: Plasma Isolation
7. Freeze at -80 °C.

SECTION: Step 2: PBMC isolation
8. Dilute the rest of the whole blood sample to a 1:1 volume ratio with the PBS
SECTION: Step 2: PBMC isolation
9. Add 15 mL density gradient medium to a fresh 50mL conical tube and gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.

SECTION: Step 2: PBMC isolation
10. Centrifuge at 800 x g, 20 min 

SECTION: Step 2: PBMC isolation
11. Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface. Alternatively, you can first remove the upper layer and then collect the cells
SECTION: Step 2: PBMC isolation
12. Wash the harvested PBMC in 12 mL of PBS

SECTION: Step 2: PBMC isolation
13. Centrifuge at 300 x g, 15 min 

SECTION: Step 2: PBMC isolation
14. Discard the surnatant and wash the PBMC pellet in 5mL of PBS
SECTION: Step 2: PBMC isolation
15. Centrifuge at 300 x g, 15 min 

SECTION: Step 2: PBMC isolation
16. Discard the surnatant and resuspend the cell pellet in 5 mL of PBS

SECTION: Step 2: PBMC isolation
17. Count the cells using Trypan blue staining.
SECTION: Step 2: PBMC isolation
18. Resuspend PBMCs to 5x106 cells/mL in PBS and aliquot them in cryotube vials (1mL/vial) labeled with patient ID
SECTION: Step 2: PBMC isolation
19. Centrifuge at 800 x g, 10 min 

SECTION: Step 2: PBMC isolation
20. Discard the surnatant and store the cell pellets at -80 °C
TITLE: Karyotype on fixed mealybug tissue
AUTHORS: Isabelle Vea, Stevie Bain

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

[STEPS]
?. Males testes:   Fix material in Bradley Carnoy (4:3:1 chloroform: ethanol: acetic acid) and keep at 4 degree celcius overnight (can keep up to 7 days for male testes)Embryos: no need to fix
?. Males testes:  Dissect the tissue in 45% acetic acid (add 20ul of 45% acetic acid on a slide coverslip, pipet out the males and place them on a filter paper, transfer the male in the acetic acid with a needle, dissect)Embryos: use freshly laid embryos and transfer directly in 45% acetic acid on coverslip
?. Use a slide to catch the coverslip by surface tensionEmbryos: reverse the slide and tap gently on the coverslip a few times so that the fixative goes in the embryos cells
?. Place a filter paper on the bench, then place the slide on top so you can fold the filter paper back to the coverslip, press  firmly with thumbs (ideally, the fixative excess should squeeze out). AVOID SLIDING COVERSLIP ON THE SLIDE
?. Dip the slide with forceps in liquid nitrogen until the nitrogen doesn't bubble anymore
?. Snap the coverslip with a razorblade
?. Add 40 uL of Ethanol:Acid Acetic 3:1 on the slide where the sample is
?. Let the slide air dry completely
?. Add vectashield DAPI
?. add a coverslip and seal with nail polish and let it incubate in a drawer for 30 min before looking at the slide
TITLE: Field Collecting Protocol for Vascular Plants. Denver Botanic Gardens. Kathryn Kalmbach Herbarium of Vascular Plants
AUTHORS: Christina Alba, Melissa Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Floristic surveys and plant collections represent baseline data critical to understanding plant species diversity and distributions. To make plant collections valuable for current and future researchers, both the physical specimen and all associated data must be of the highest quality. This protocol describes the steps by which researchers at Denver Botanic Gardens make vascular plant specimen collections and record associated data.</div></div>

[STEPS]
TITLE: Mouse Cardiac Perfusion Fixation and Brain Collection
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the procedures for intracardiac perfusion fixation of postnatal mice, including anesthesia, exsanguination, fixation, brain removal and post-fixation storage. 

Note: 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.

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>1. Many factors can affect blood pressure (BP) measurements: first-time measurements, temperature, stress, noise and inappropriate handling.</p>
<p>2. Measure a minimum of 3 days and a maximum of 5 consecutive days.</p>
<p>3. Measure at the same time each day because BP varies according to the time of day (circadian rhythm).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Biochemical Measures of Neuropathy - Aconitase
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biu44kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation — Tissue]
1. Weigh tissue sample. 2. Mince tissue. 3. Homogenize at 1% (w/v) in ice cold 0.2mM sodium citrate for 15-20 seconds. (new protocol calls for sodium citrate in 50mM Tris-HCl, pH 7.4). 4. Centrifuge at 800 x g for 10 minutes at 4°C. 5. Remove 25µl supernatant for protein assay. 6. Remove supernatant and store on ice or freeze –80°C until use.
?. [Performing Assay:]
1. Thaw homogenate on ice. 2. Fill in layout on computer and save as acxxxxxx.sed where xxxxxx is the date in yyddmm format. 3. Sonicate for 20 seconds. 4. Dilute the tissue extract 10µl extract into 190µl (500µg/ml) assay buffer. 5. Add 50µl of sample to each well in triplicate. (Do 1:1 and 1:20 dilutions.) 6. Add 50µl assay buffer to blank well in triplicate. 7. Add 50µl of Substrate to each well. 8. Add 50µl of Enzyme to each well. 9. Add 50µl of NADP to each well. 10. Mix using a pipet
?. [Reading the Plate:]
1. Record absorbance change at 340nm for 5 minutes at 37ºC. 2. Turn on Multiskan and open your saved file from above. 3. Place plate onto Multiskan holder and click START. 4. Save raw data as an Excel file into the acx data folder. Use the naming convention acXXXX.xls, where XXXX is the date in mmdd format. 5. Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns. 6. Save organized data as an Excel file into the acx data folder. Use the naming convention acXXXXor.xls, where XXXX is the date in mmdd format.
TITLE: Fixation of Eyes at UAB
AUTHORS: David       Anderson, Jeffrey     Messinger, Angela Kruse, Jamie Allen, Carrie  Romer, Kevin      Schey, Jeff Spraggins, Christine      Curcio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The accession and fixation of whole human eye specimens at University of Alabama Birmingham.</div></div>

[STEPS]
?. Whole eyes arrive from deceased human donors via Advancing Sight Network (Birmingham AL; formerly the Alabama Eye Bank) ≤6 hours death-to-preservation.
?. Fixation is performed on the globe with lens and iris in place by immersing in buffered 4% paraformaldehyde overnight.
?. Initially the cornea is removed with a 2-mm-wide scleral rim boundry, a circular cut is scored with an 18-mm trephine (Stratis Healthcare, #6718L) and completed with curved spring scissors (Roboz Cat# RS-5681).
?. To facilitate preservative penetration, without disturbing the vitreous body and detaching the retina from RPE, the iris is slit with a small incision.
?. The iris and lens were removed before imaging with OCT and scanning laser ophthalmoscopy (Anderson et al., 2020).
TITLE: Western blotting of Syp-VAMP2 complex
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context impeding  our ability to quantitatively decipher the local molecular context. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome enabling near-complete spatially-resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from any endogenous organellar membrane at physiological expression levels. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane ‘nano-scoops’ containing a target MP and interface with structural, functional, and other bioanalytical approaches. 

[STEPS]
SECTION: Reagents needed for protocol
1. Acquire the following antibodies for the Western blotting protocol:
SECTION: Reagents needed for protocol
1.1. Primary Antibody: rabbit Na/K ATPase (Abcam Cat# ab167390, 1:5000)
SECTION: Reagents needed for protocol
1.2. Primary Antibody: Synaptophysin Rabbit mAb (ABClonal Cat#A19122, 1:5000)
SECTION: Reagents needed for protocol
1.3. Primary Antibody: VAMP2 Rabbit mAb (ABClonal Cat#A4235, 1:5000)
SECTION: Reagents needed for protocol
1.4. Secondary Antibody: HRP Goat Anti-Rabbit IgG (H+L) (ABClonal Cat#AS014, 1:10000)
SECTION: Running the gel and transfer
3. Run the gel until the loading dye front runs off the ends of the gel.
SECTION: Running the gel and transfer
2. Measure protein concentration with Bradford Kit, dilute into 4x Laemmli buffer to bring final concentration to 1x, and load to a 4-20%Mini-PROTEAN TGX precast gel.
SECTION: Running the gel and transfer
4. Using a TurboBlot system, transfer gel onto a PVDF membrane.
SECTION: Blotting
5. Block membrane in 5% milk for 60 min and wash with TBST

SECTION: Blotting
6. Incubate with the primary antibody at the appropriate concentration listed above  at 4 °C  

SECTION: Blotting
7. Wash with TBST
SECTION: Blotting
8. Incubate with the secondary antibody at the concentration listed above for 60 min  and wash with TBST

SECTION: Blotting
9. Add HRP and detect chemiluminescence using an ImageQuant system.
TITLE: Protocol for hippocampal neuronal cultures
AUTHORS: Andrés Guillén-Samander, Pietro De Camilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details the procedure for preparation of neuronal cultures from mice hippocampi as it was performed in </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1083/jcb.202010004</span></a><span style = ":;:UNDERLINE;"> </span><span>but can also be used to prepare cultures of cortical neurons.</span></div></div>

[STEPS]
?. [Protocol for hippocampal neuronal cultures]
Coat MatTek dishes with  per dish of  Poly-D-Lysine (Sigma) for at least  to  at .
1 mL
37 °C
?. [Protocol for hippocampal neuronal cultures]
Wash dishes twice with culture grade water and let dry.
?. [Protocol for hippocampal neuronal cultures]
Prepare papain solution and leave at  for .
37 °C
?. [Protocol for hippocampal neuronal cultures]
Dissect hippocampi from at least 3 P0 mouse brains using a stereo microscope. Collect tissue in ice cold Neuronal HBSS (nHBSS).
?. [Protocol for hippocampal neuronal cultures]
Transfer isolated hippocampi into a fresh cold nHBSS containing dish.
?. [Protocol for hippocampal neuronal cultures]
Cut tissue into ≈1mm3 pieces and transfer into a  Falcon tube with  ice cold nHBSS and let sediment .
15 mL
10 mL
on ice
Note: From this step on everything is done under a sterile hood.
?. [Protocol for hippocampal neuronal cultures]
Aspirate medium and wash 2-3 times with  fresh ice cold nHBSS.
10 mL
?. [Protocol for hippocampal neuronal cultures]
Add DNAse to papain solution and filter sterilize. Incubate tissue prep with papain solution for  at  on a rocking platform.
37 °C
?. [Protocol for hippocampal neuronal cultures]
Aspirate the enzyme solution and wash twice with plating medium and then twice with nHBSS.
?. [Protocol for hippocampal neuronal cultures]
Allow debris to settle for several minutes and collect supernatant.
Warning: Tissue will be softer after papain solution, handle with care to avoid dissociating the cells.
?. [Protocol for hippocampal neuronal cultures]
Resuspend samples in  cold nHBSS. Gently dissociate neurons with a P1000 filter tip by pipetting up and down for 10-12 times.
2 mL
Warning: Avoid generating any bubbles.
?. [Protocol for hippocampal neuronal cultures]
Count the cells.
?. [Protocol for hippocampal neuronal cultures]
For imaging, seed 75,000 neuronal cells as a drop (usually around ) in the PDLcoated coverslip of MatTek dishes in Plating Medium.
100 µl
?. [Protocol for hippocampal neuronal cultures]
After  to  incubate at  and 5% CO2, change the plating medium to neuronal medium.
37 °C
?. [Protocol for hippocampal neuronal cultures]
Remove  of media and add  of fresh neuronal media every 3-4 days.
500 µl
1 mL
Suggestion: Incubate the media at   and 5% CO2 before adding it to neurons.