eamag/protocols-with-steps huggingface dataset:
TITLE: Biospecimen Collection Protocol
AUTHORS: Creative Bioarray

[DESCRIPTION]
A wide variety of specimen types may be collected for storage, depending on availability and study goals, such as tissues that from autopsy and transplant, cell lines, and so on.

[GUIDELINES]
A wide variety of specimen types may be collected for storage, depending on availability and study goals, such as tissues that from autopsy and transplant, cell lines, and so on.
Timing. In general, it is important to minimize the time between collection and stabilization and processing of tissue specimens. This time will vary according to the intended use, since different biomolecules degrade at different rates. The effects of collection timing on tissue and macromolecule preservation have not been well studied. The best approach is to collect, stabilize (freezing or fixing) and process tissue specimens as rapidly as possible.
Surgical specimens. Remnant samples may be collected from diagnostic procedures. Depending on the intended use, specimens may be transported or frozen immediately. Samples requiring snap freezing can be frozen in a Dewar flask of liquid nitrogen or on dry ice at the time of collection. Otherwise, it is recommended that samples be transported in saline on wet ice to the repository or laboratory for additional processing.
Transplant tissue and organs that are inappropriate for transplant may sometimes be made available for research. Often transplant tissue is of a higher quality than either surgical or autopsy specimens, due to the special efforts made to preserve the integrity of the transplant organs.

[STEPS]
1. Serum. Coagulate whole blood at room temperature (20-25°C) for 20-30 min; 1,000 x g at room temperature for 10 min; then collect supernatant, or optionally use a serum separator. If the sample is hyperlipidemic, first centrifuge at 2-8°C 10,000 x g for 10 min to separate out the lower serum layer.
Plasma. Choose citric acid or EDTA as anticoagulant according to the requirements. If heparin is chosen as the anticoagulant, it is recommended that no more than 10 IU of heparin per ml of blood be used (too high a dosage of heparin may result in high values for some assays). Within 30 min after collection of anticoagulated whole blood, the plasma is separated and collected by centrifugation at 10,000 x g at 4°C for 10 min. If the sample is hyperlipidemic, first centrifuge at 2-8°C at 10,000 x g for 10 min to separate the lower plasma layer.
Tissue samples. Specimens are rapidly frozen in liquid nitrogen for storage after removal. Keep the specimens at 2-8°C even after thawing. Add an amount of buffer containing protease inhibitors and homogenize the specimens by hand or with a homogenizer. Centrifuge for about 20 min (2000-3000 rpm) and carefully collect the supernatant. If precipitate is present during storage, the sample should be centrifuged again to remove the precipitate before the assay. 16,000 x g centrifugation for 5 min is recommended.
TITLE: Toehold testing/ PURE system protein production
AUTHORS: Junyan Qian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Our Toehold switches are tested using NEB PURExpress and OnePot PURE system (our own cell-free protein expression system, search OnePot for detailed protocols). Our goal is to define a concentration of Toehold switch and trigger DNA sequence which has a high protein (sfGFP in our case )expression but low or no leakage at same time.</div></div>

[STEPS]
?. Take a clean 384-well micro plate, mark the using wells on the plastic cover, when finished put the cover underneath the micro plate, the marks should be seen though the wells, so it reduces the risk of miss pipetting.
?. Make OnePot premix, an supplemental step if using OnePot system and having experiments more than 5 reactions. Energy solution has to keep separately all the time. The OnePot energy solution and OnePot premix reaction volume is referring to the NEB PURExpress solution A  and B accordingly in following steps.
?. OnePot protein solution : OnePot ribosome solution = 13 : 18. (The concentration of the solution is indicated in the OnePot protocol)For a   reaction a   of premix is needed. (exp. 5 x   premix contains  of OnePot protein solution and   of  OnePot ribosome solution )Tips: when making the premix, always make one extra reaction volume to make sure that in the end, there won't be short of premix solution.
5 µl
1.5 µl
1.5 µl
4.64 µl
6.43 µl
?. There is limited space in a  reaction, after solution A and solution B there will be only  space for both toehold and trigger. It's better to dilute or concentrate them to the desired concentration before adding. The following steps are referring to a  of toehold switch and  of trigger dsDNA as an example.
5 µl
1.5 µl
?. Pipetting:  ABCDEFGH1Reference/rxn concentration (5μL)Toehold_1 (+)Toehold_2 (+)Toehold_3 (+)Toehold_1 (-)Toehold_2 (-)Toehold_3 (-)2H2Ofill till 5 μL///1113A/Energy22222224B/Premix1.51.51.51.51.51.51.55Toehold_15nM0.5//0.5//6Toehold_25nM/0.5//0.5/7Toehold_35nM//0.5//0.58BN_trigger2uM111///9RNAse inhibitor0.20.20.20.20.20.20.210Total:5555555Order of adding the reagents: Solution A (Energy solution) + water -> Solution B (OnePot premix) -> DNA template
ABCDEFGH1Reference/rxn concentration (5μL)Toehold_1 (+)Toehold_2 (+)Toehold_3 (+)Toehold_1 (-)Toehold_2 (-)Toehold_3 (-)2H2Ofill till 5 μL///1113A/Energy22222224B/Premix1.51.51.51.51.51.51.55Toehold_15nM0.5//0.5//6Toehold_25nM/0.5//0.5/7Toehold_35nM//0.5//0.58BN_trigger2uM111///9RNAse inhibitor0.20.20.20.20.20.20.210Total:5555555
?. Balance the centrifuge,  .
Centrifuge: 4000 33
?. Add  of BIO-RAD Chillout liquid wax to each well. Place the wells gently inside the plate reader, check the setting of the plate reader, start the measurement.
35 µl
?. When the measurement is done, export the data.
TITLE: gDNA Extraction with QuickExtract
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This uses the Quick Extract solution to rapidly obtain gDNA. </div><div class = "text-block">I found this protocol on-line written by Patrick Hsu but can no longer find the original link. The protocol is nice because it does not require an extra vortexing step. It has worked well for hundreds of gDNA extracts. </div><div class = "text-block">For quick work I often pellet an uncounted small amount of cells and use 30ul QE and it has always worked. This has worked for me with mouse/human T and B cells, iPSCs, HeLa, K562. </div></div>

[STEPS]
?. [Pellet Cells]
Count cells if you want gDNA concentration.The quick extract interferes with the nanodrop and so quantification of gDNA is easier by adding a known volume to a known about of cells. 2N human cell = 6 pg gDNA-> 100ng is 16,000 cells.  (32k alleles)For 100ul of 50ng/ul:  8,000,000 cells  re-suspend in 100ul QuickExtract (this will give you roughly the right concentration)Normally for quick genotyping I just pellet some cells then resuspend in 20-50ul QE. I do PCR with 35x cycles (PrimeStar GXL is best polymerase). This has always worked and is very low energy required.Count cells and then pellet the desired amount in 96 well plate (2000rpm 2 min).
?. [Pellet Cells]
Wash once with PBS (or just tap dry). Then re-pellet if you did a wash.Resus in QE, pipette up and down to mix, transfer to PCR strip.*Often I don't wash and this hasn't seemed to create an issue during PCR. *
?. [Pellet Cells]
To extract the gDNA from the cells:Put on thermocyclcer65C for 15 min68C for 15 min98C for 10 minHold at 4C*I have left at 4C for months and things work fine. Also I have frozen and thawed and it works fine.
TITLE: Modified protocol to improve Bodo saltans yield in culture.
AUTHORS: Zhu-Hong Li, Fatma Gomaa, Virginia Edgcomb, Roberto Docampo

[DESCRIPTION]
This protocol is a modified version to improve the yield of Bodo saltans cell density in culture. The original protocol is: https://www.protocols.io/view/bodo-saltans-culture-protocol-sh6eb9e

[STEPS]
1. Prepare the bacteria-Bodo saltans medium as described in the protocol （https://www.protocols.io/view/bodo-saltans-culture-protocol-sh6eb9e）. 
2. Collect Bodo cells from a T25 tissue culture flask by centrifugation at 1200 x g for 6 minutes. After removing the supernatant, add deionized water to resuspend the pelleted cells. After two washes with water, resuspend the cells in culture medium. Usually the Bodo saltans/bacteria ratio is  >1 after two washes, as determined by FACS analysis. 
3. Inoculate a T25 tissue culture flask (50 ml) containing 10 to 15 ml of fresh medium with 10 mg/ml puromycin. Puromycin at this concentration has no inhibitory effect on the growth of Bodo saltans but it can slow down bacterial growth. Transfer 100 ml-0.5 ml cells from step 2 into the flask.  Incubate horizontally at 18C with loosely adjusted cap.
4. After 2-4 days, when the density of Bodo cells reaches to ~3 X 106/ml, collect and wash the cells for downstream applications.
5. The advantages of this protocol are improved yield and purity. 10 mg/ml puromycin can efficiently prevent bacterial population overgrowth in the flask. The medium is much cleaner and there are no aggregates formed by overgrown bacteria. Usually, we can only get 1 X 106/ml of Bodo cells using conventional culture conditions, and need filtration and 4-5 washes to remove all bacteria from the culture for other applications, such as electroporation or DNA extraction. After these modifications Bodo cells can reach 3 X 106/ml in the culture resulting in a purer population of cells after only 2 washes.
6. {"blocks":null,"entityMap":null}
TITLE: Sanger Tree of Life Wet Laboratory Protocol Collection 
AUTHORS: Amy Denton, Halyna Yatsenko, Jessie Jay, kh, Caroline Howard

[DESCRIPTION]
This is the collection of Sanger Tree of Life Wet Laboratory protocols used for the generation of reference level genome assemblies. 

This collection currently contains protocols which cover all stages of the wet laboratory processes performed by the Sanger Tree of Life; these processes are sample preparation, sample homogenisation, HMW DNA extraction, HMW DNA fragmentation, fragmented DNA clean-up and RNA extraction.

This collection will be updated as protocols within the Tree of Life Core Laboratory continue to be developed and refined.


  


[STEPS]
TITLE: Seedling vigor
AUTHORS: Zhenbin Hu, Geoff Morris

[DESCRIPTION]
<ul>
<li>Select five plots as standard: 1 (weakest) - 5 (strongest), then other plots will be scored based on the similar with which standard plot.</li>
<li>Seedling vigor is based on the size of seedling, greenness, uniformity</li>
<li>Score each plot for seeding vigor was phenotyped by plot</li>
<li>Seedling vigor was phenotyped every other day for six times</li>
<li>Seedling vigor phenotyping was started after 15 days of planting or 10 days after emergence</li>
</ul>

[STEPS]
?. [Identify Standard Plots]
Go through the field and identify five plots to use as standards that represent scores ranging from 1 to 5
?. [Visit each plot]
Go through the field to do seedling vigor phenotyping by compare the seedling with the standard plot
?. [Score Plants]
Assign a score between 1 and 5 to each plot using the scale developed from standard plots
TITLE: Isolation of peripheral blood mononuclear cells
AUTHORS: Devi Oktafiani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and  monocytes. PBMCs are separated from the whole blood by a density  gradient centrifugation method using Ficoll-Paque.</div></div>

[STEPS]
?. Freshly blood collected in EDTA tube
?. Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771; Sterile PBS
?. Collect 4 ml of human venous blood sample in EDTA tube and mix well by gently inverting the tube several times.
?. Take 5 ml of Ficoll Histopaque in a 15 ml centrifuge tube.
?. Gently layer the blood on the top of Ficoll Histopaque using a 1 ml auto pipette. The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers.
?. Centrifuge the tubes (without any delay) for 30 min at 100 x g in 4 °C in a swing-out bucket. Fixed angle rotors also can be used but would require more caution when separating cells in interphase.
?. Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium.
?. The cells in interphase need to be aspirated without delay. If the tubes are kept standing for more than 10 min, PBMCs from the interphase will get disturbed and start settling down.
?. Wash (centrifuge in 100 x g for 10 min) twice with 10 ml of sterile PBS
?. Remove PBS
?. Add PBS 1 mL and transfer (PBS and cells) into 1,5 mL new tube
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How we measure the gradient of he density of 19th century post offices along North American rivers in our paper Armenter, Roc, Miklós Koren and Dávid K Nagy. “Bridges” 

Method relies on maximum likelihood formula in Zhao, M., and M. Xie. 1996. “On Maximum Likelihood Estimation for a General Non-Homogeneous Poisson Process.” Scandinavian Journal of Statistics, Theory and Applications 23 (4): 597–607.

[STEPS]
SECTION: Select relevant sample of points close to linear feature
?. 
SECTION: Project points on linear feature and convert to linear coordinates
?. 
SECTION: For each point, measure distance to nearest crossing point
?. 
SECTION: Given the distribution of distances, estimate gradient parameters by maximum likelihood
?. 
SECTION: Convert all features to a distance-invariant projection
?.
TITLE: Production of AAV Virus by Iodixanol Gradient Ultracentrifugation
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to produce AAV of any serotype with high purity and high titer.

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: Anti-BrdU Staining Using 70% Ethanol and 2N HCl
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Note: We offer two protocols here depending on what your experiment requires. Ethanol treatment is usually harsher toward any other fluors or fluorescent proteins that may be present in your sample. As such, the DNAse method may be gentler under those conditions</div></div>

[STEPS]
?. Pulse actively dividing cells with BrdU (in vitro, cell culture media can be pulsed by adding 10-40µM of BrdU for 1-2 hours).
?. Harvest cells and centrifuge for 5 minutes at 1200-1500 rpm (200-300xg).
?. Wash cells in 1x PBS (PBS, 10x Concentrate, Cat. No. 926201) and centrifuge for 5 minutes at 1200-1500 rpm (200-300xg). Discard supernatant.Note: The combined presence of proteins and HCl in downstream steps may cause aggregation. As such, it is highly recommended that wash steps utilize PBS without any protein additive until otherwise indicated.
?. Dislodge cell pellet and add 5ml of ice-cold (-20°C) 70% Ethanol to 1-2 X 107 cells dropwise while slowly vortexing. Incubate at -20°C for at least 2 hours. Cells may be stored for several days.Note: For ethanol permeabilization, we recommend this step last for at least 2 hours as this improves permeabilization and overall staining. If you’re still having difficulty, you can attempt to extend this to an overnight incubation (assuming you aren’t also staining for cell surface markers that could be lost).
?. Repeat step 3 twice.
?. Dislodge cell pellet and add 2ml of 2 N HCl and incubate for 20 minutes at room temperature.
?. Repeat step 3.
?. Dislodge cell pellet and add 2ml of 0.1M Na2B4O7 for 10 minutes at room temperature.
?. Repeat step 3.
?. Resuspend cells at a concentration of 1 x 107 cells per/ml of staining buffer and aliquot 100µl per tube. Add anti-BrdU antibody at appropriate concentration and incubate for 20 minutes at room temperature.
?. Wash cells in Cell Staining Buffer (Cat. No. 420201) and centrifuge for 5 minutes at 1200-1500 rpm (200-300xg).
?. Stain DNA by adding 1µg of either 7-AAD (Cat. No. 420403) or DAPI (Cat. No. 422801). Wait for 5 minutes prior to acquiring samples on flow cytometer.Note: Adding a 7-AAD or DAPI stain allows you to analyze total DNA content and provides the characteristic horseshoe flow cytometric staining pattern when compared against BrdU. This helps identify the different phases of the cell cycle.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Autodigest Proteinase K, 25 mg/mL, at 37°C 60 min prior to use<br />Heat RNAse A, 10 mg/mL, at 80°C 10 min prior to use

[GUIDELINES]
<strong>MATERIALS:</strong> <br /><br /> 1) NC64A chlorella<br /> 2) Sterile 500 mL flasks<br /> 3) MBBM<br /> 4) 50 mM NaHPO<sub>4</sub>, pH 7.4, 2.0 M NaCl<br /> 5) 20% SDS<br /> 6) Proteinase K, 25 mg/mL, autodigested at 37°C, 60 min prior to use<br /> 7) RNAse A, 10 mg/mL, heated at 80°C, 10 min prior to use<br /> 8) 5 M KOAc<br /> 9) 3 M NaOAc<br /> 10) 100% EtOH<br /> 11) 70% EtOH<br /> 12) Isopropanol<br /> 13) 50 mM Tris-HCl, pH 8.0, 10 mM EDTA<br /> 14) 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (1X TE)<br /> 15) Sterile d-H<sub>2</sub>O

[STEPS]
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TITLE: Live-cell imaging: Reactive oxygen species (Superoxide)
AUTHORS: gurvir.virdi, mineechoi

[DESCRIPTION]
This protocol contains the instruction for measuring superoxide using dihydroethidium (HEt) which allows the rate of superoxide generation to be measured which is present as the ratio of the oxidized form of the dye over the reduced form. HEt allows the rate of cytosolic superoxide generation to be measured, by the slope of the ratio of oxidized dye to reduced dye.

[STEPS]
1. Cells are washed 1x in HBSS 
2. They are then incubated with  2 µM dihydroethidium (HEt; Thermo Fisher Scientific) made up in HBSS.
 

3. Immediately after adding HEt, a field of view is found and confocal microscopy is started.
4. The recording is performed using an epi-fluorescence inverted microscope equipped with 20x objective after a quick loading (2-3 min) in order to limit the intracellular accumulation of oxidised product. Throughout imaging, the dye is present in the buffer. 
4.1. Live-cell imaging is performed using an epi-fluorescence inverted microscope equipped with a CCD camera (Retiga; QImaging). For epi-fluorescence inverted microscope, excitation is provided by a xenon arc lamp with the beam passing through a monochromator (Cairn Research) and emission was reflected through a long-pass filter to a cooled CCD camera and digitized to 12-bit resolution (Digital Pixel Ltd, UK).
4.2. A time-series with 5-10 second intervals is performed. 

Excitation is at 530 nm, and emission recorded above 560 nm is assigned to the oxidized form, while excitation at 380 nm and emission collected from 405 nm to 470 nm is assigned to the reduced form. 
4.3. The ratio of the fluorescence intensity, resulting from its oxidized/reduced forms, is quantified.
TITLE: Chlorophyll Extraction in Cyanobacteria
AUTHORS: Nicolas Schmelling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol should be used for chlorophyll extraction in cyanobacteria. The equation for calculating the exact chlorophyll content can be found at the end of this document.</div><div class = "text-block">You might want to measure the optical density (OD) of you cyanobacteria culture at 750 nm. Use BG11 medium or water as the reference solution. You need the OD of your culture to normalize the cholorphyll concentration to the number of cyanobacteria.</div><div class = "text-block"><span style = "font-weight:bold;">Calculate chlorophyll content </span><span>(adapted from </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Lichtenthaler 1978</span></a><span style = ":;">)</span></div><div class = "text-block"><span>Chl [µg/ml] = OD</span><span style = "vertical-align:sub;">665nm</span><span> x 13.9 [µg/ml] x dilution factor of culture</span></div><div class = "text-block">You can take less than 1 ml, but note the dilution factor for the calculation later on, e.g. :</div><div class = "text-block">1 ml sample = dilution factor of 1</div><div class = "text-block">500 µl sample =  dilution factor of 2</div><div class = "text-block">100 µl sample = dilution factor of 10</div></div>

[STEPS]
?. Take 1 ml sample of your cyanobacteria culture and spin it down at 14,000 rpm for 5 min.
?. Discard 0.9 ml of the supernatant. Resuspend the pellet in the remaining 100 µl.
?. Add 0.9 ml of 100% methanol to the sample and mix thoroughly by vortexing.
?. Incubate the samples in the dark for 30 min at 4 °C in the fridge.
?. Spin down samples again at 14,000 rpm for 5 min.
?. Transfer supernatant into a cuvette and measure the extinction at 665 nm. Use 90% methanol as the reference solution.
TITLE: Latest Instagram Marketing Techniques For 2021
AUTHORS: Amit kumar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Instagram began as an image-sharing website. Now it is a huge social media platform that can help businesses get more leads and generate more revenue.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">They say, "Change with the trend!"Instagram marketing, like other social media sites, has evolved over time. It's all about being more interactive with your followers!</div></div></div>

[STEPS]
TITLE: Yale - Magnesium
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of magnesium in blood, serum, and plasma. Magnesium is measured as the chlorophosphonazo III complex (addition of EGTA prevents calcium interference) and monitored at 600 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Magnesium analysis by running the Magnesium Calibrator and a Water Blank.
?. Sample handling as performed by the Cobas Mira Plus    a) Pipette 4 µL of sample into a cuvette slot.    b) Add 350 µL of Magnesium Reagent.    c) Mixture is incubated at 37ºC for 10 minutes    d) Absorbance is measured at 600 nm.