eamag/protocols-with-steps huggingface dataset:
TITLE: Restriction Digest of Plasmid DNA
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for restriction digest of plasmid DNA. To see the full abstract and additional resources, please visit </span><a href="http://www.addgene.org/plasmid_protocols/restriction_digest" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.addgene.org/plasmid_protocols/restriction_digest/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Select restriction enzymes to digest your plasmid.
Notes:For a list of many commonly used restriction enzymes, visit NEB.To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer.
?. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
?. In a 1.5mL tube combine the following (typically  reaction): DNA (all amounts are for a typical reaction; your amount may vary depending on the enzymes)  of each restriction enzyme of Bufferof BSA (if recommended by manufacturer)dH2O up to total volume (up to for typical reaction)
50 µl
1 µg
Notes:The amount of DNA that you cut depends on your application. Diagnostic digests typically involve ~  of DNA, while molecular cloning often requires  -  of DNA. The total reaction volume usually varies from  -  depending on application and is largely determined by the volume of DNA to be cut.See the Tips and FAQ in the guidelines section for determining what volume to use for restriction enzymes.
1 µl
Restriction enzymes MUST be placed in an ice bucket immediately after removal from the   freezer because heat can cause the enzymes to denature and lose their function.
3 µl
3 µl
30 µl
?. Mix gently by pipetting.
?. Incubate tube at appropriate temperature (usually ) for .
37 °C
*Pro-Tips*Depending on the application and the amount of DNA in the reaction, incubation time can range from   to overnight. For diagnostic digests,  -  is often sufficient. For digests with >  of DNA used for cloning, it is recommended to digest for at least  .If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction. This may involve incubating the reaction at   for  , or purifying the DNA via a purification kit, such as a QIAGEN DNA cleanup kit. See the enzyme manufacturer's instructions for more details.
To visualize the results of your digest, conduct gel electrophoresis.
TITLE: Hybridization of Soybean Via Cross-Pollination
AUTHORS: Lynn   Doran

[DESCRIPTION]
Cross-pollination of soybean to hybridize two lines, introgress alleles, or transfer a transgenic event to a new genetic background.

https://www.youtube.com/watch?v=-iJXSPzKZO0
https://www.youtube.com/watch?v=VnjGijF4KQI
https://www.youtube.com/watch?v=BEe6b8tdf_8

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the tracing of newly deposited silica using PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole) in reproductive studies of diatoms.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Ancient DNA extraction  (chunk samples/high volume)
AUTHORS: Marcel Keller, Christiana L Scheib

[DESCRIPTION]
Extraction protocol for ultra-short ancient DNA molecules from skeletal chunk samples modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).

[BEFORE_START]
Previous steps:
This protocol follows the sampling of chunk samples (tooth roots, petrous cores, other bones) and decontamination. Step 2 assumes that the samples have been dried under UV light in the laminar flow hood (see decontamination). Alternatively, the samples can be transferred into tubes.

Following step:
This protocol ends with a 72 h incubation step. Proceed with the purification (chunk samples/high volume) after the incubation.

Equipment and consumables:
 



[GUIDELINES]
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.

[STEPS]
SECTION: Extraction
5. Clean table bench surface with DNA Exitus and wipe dry. Turn the hood on full power and open the glass.
SECTION: Extraction
6. 
 
Move dry samples from the hood to the table bench and put down a new sheet of aluminum foil.

SECTION: Extraction
7. Clean hood surface with DNA Exitus and put down a new sheet of aluminum foil. Wipe down reagents with DNA Exitus.
SECTION: Extraction
8. Place all reagents (except proteinase K) and consumables in the hood and UV while weighing.
SECTION: Extraction
9. 
 
Tare scale with small weighing boat labeled with the sample number, place tooth/petrous core on the weighing boat with the tweezers and record weight. Change gloves when finished with weighing.
SECTION: Extraction
10. Make EDTA 0.5 Molarity (M)pH 8.0, Proteinase K 18.18 mg/mL, and tube calculations:


 

SECTION: Extraction
11. Label your tubes for the extractions:

 

 
 
SECTION: Extraction
13. Add calculated amount of EDTA to each tube. If the tubes are empty you can use the same pipette tip as long as you do not get liquid into the filter or touch any surfaces with the tip.
SECTION: Extraction
12. If necessary: Make Proteinase K solution by adding  5.5 mL PCR clean water to 100 mg of powder. Shake to dissolve. Let sit before using. Store in the freezer at -20 °C.

SECTION: Extraction
14. Add the calculated amount of Proteinase K to each tube and pipette-mix. Change your tips each time.
SECTION: Extraction
15. Add samples to their individual extraction tube. 
SECTION: Extraction
16. Make sure that the lids are tightly sealed to avoid leakage.
SECTION: Extraction
17. Wipe tubes with DNA Exitus, wrap tubes with Parafilm.
SECTION: Extraction
18. Place on the nutating mixer for  .

 


SECTION: Prerequirements
1. 
 



SECTION: Prerequirements
2. 
 

SECTION: Extraction
3. Set up decontamination station: 
4 disposable 100 ml beakers
6% NaOCl (bleach) aliquot [2x] 
MilliQ water [1x]  
70% ethanol aliquot [1x]
SECTION: Extraction
4. To decontaminate tweezers, soak in 6% bleach for 5 minutes, rinse with MilliQ water, rinse with 70% Ethanol.
TITLE: Testing and validation of a Patient-Reported Outcome Measure for Older People with frailty and Acute Care needs (PROM-OPAC).
AUTHORS: James D van Oppen

[DESCRIPTION]
Aim: To refine, validate, and evaluate the early impact of a novel PROM instrument, the Patient-Reported Outcome Measure for Older People with frailty and Acute Care Needs (PROM-OPAC).

[STEPS]
SECTION: Preparatory work
1. Semi-structured interviews with 15-30 older people living with frailty with recent experience of UK emergency and acute care.
Qualitative analysis using blended approach of framework and comparative methods.
Outcome: a framework to define outcome goals for acute healthcare among older people living with frailty.
SECTION: Preparatory work
2. Scoping review to identify existing patient-reported outcome measure instruments with relevance and potential validity to older people with frailty in the acute care setting.
SECTION: Preparatory work
3. Development of a novel patient-reported outcome measure for this setting.
Review of existing instruments and extensive comparison and reflection with the framework of health outcome goals which has been produced, in order to prepare and exhaustive list of item stems, questions, and responses.
Reduction, iterative refinement, and evaluation of content validity of the exhaustive list through patient review in interviews, lay collaborator review in surveys, and clinician review in surveys and short interviews, in order to prepare a draft PROM instrument.
SECTION: Stage 1
4. Field-testing the draft instrument in UK sites with a representative sample of 150 older people living with frailty.
Evaluation will include feasibility in the form of response rates and feedback, internal consistency, and retest reproducibility, and patient feedback to inform iterative development.
Outcomes will be reviewed with lay collaborators and clinicians.
SECTION: Stage 2
5. Disseminated testing and validation in separate sites with a further sample of 150 older people living with frailty.
Evaluation will include interpretability and construct validity through comparison with alternative measures of healthcare quality.
SECTION: Stage 3
6. Evaluation of early implementation and potential applications for PROM-OPAC using follow-up interviews with a small sample of Stage 1 & 2 participants.
TITLE: Serum, Blood and Body Weight Measurements in Pigs
AUTHORS: Tim Nichols, Dave Clemmons

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">These assays are performed on all the pig models studied by the DiaComp to assess their serum chemistry and blood cell counts.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiqe.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiqn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiqw.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiq6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakire.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakirn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakirw.jpg" /></div></div>

[STEPS]
?. Total cholesterol
Cholesterol is measured by a commercial, automated method (ANTECH® Inc. Cary, NC).
?. Serum assays
All serum assays are measured by a commercial, automated method (ANTECH® Inc. Cary, NC).
?. Red blood cell count, White blood cell count, Hemoglobin, Hematocrit, Platelet count
White blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count are performed on EDTA anti-coagulated blood with a cell counter (Heska) calibrated for pig cells.
?. Body weight
Pigs are weighed on calibrated scales.
?. Percent body fat
The percent body fat is determined from measuring the backfat by ultrasound (Fukuda Denshi, model UF750XT) or the Swine Backfat Probe (Nasco, WI) according to the manufacturer’s instructions.
TITLE: Quality of COVID-19 research in preprints: a meta-epidemiological study protocol
AUTHORS: Yuki Kataoka, Shiho Oide, Takashi Ariie, Yasushi Tsujimoto, Toshi A. Furukawa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background:</div><div class = "text-block">No studies have comprehensively assessed the quality of studies related to COVID-19 in preprints [11]. Hence, we will conduct a meta-epidemiological study based on the following research question: Are the quality of COVID-19 RCT and SR articles in medRxiv lower than those indexed in PubMed? </div><div class = "text-block">Methods:</div><div class = "text-block"><span>We will conduct a meta-epidemiological cross-sectional study. We will include randomized controlled trial articles and systematic review articles indexed in PubMed or MedRxiv from 1</span><span style = "vertical-align:super;">st</span><span> January to 15</span><span style = "vertical-align:super;">th</span><span> June 2020. We will include articles of COVID-19 patients or healthcare workers engaged in the care of COVID-19 patients. We will exclude study protocols. We will evaluate the characteristics of included studies, methodological quality, number of mentions on social networking sites (SNS), and citations. We will use chi-squared test, logistic regression analysis, and linear regression analysis as appropriate. Two-tailed p values will be considered statistically significant if less than 0.05. </span></div><div class = "text-block"><div class = "justify" style = "text-align:left">Ethical consideration:</div></div><div class = "text-block">We will only use openly available data. There is no need to make ethical considerations.</div></div>

[STEPS]
TITLE: Measuring neutral lipids in fixed diatom cells using BODIPY 505/515
AUTHORS: Phoebe Argyle, Jana Hinners, Nathan G. Walworth , Sinéad Collins, Naomi M. Levine, Martina A. Doblin

[DESCRIPTION]
This protocol is for the measuring neutral lipids in fixed diatom cells using BODIPY 505/515, as developed on the centric diatom Thalassiosira spp.

This protocol has been used in the following publications:

Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes.ISME Communications,1(1), 59.

Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae.Frontiers in microbiology,12, 706235.

[STEPS]
SECTION: Diatom sample preparation
2. Sample the diatom culture of interest. For 'tube mode' flow cytometry take minimum 500 µL into an eppendorf tube or other flow cytometry tube. For plate mode take 200 µL  into a round-bottom flow cytometry plate.

SECTION: Diatom sample preparation
3. If using live cells, go to step 6

If using fixed cells:

Add 8 % volume  at  10 % (v/v)  to reach a final concentration of approx 0.8 % volume . For example, add 100 µL  of   to 1000 µL of microalgae sample.


SECTION: Diatom sample preparation
4. Agitate to mix, then leave to fix for 10 min  to ensure fixation

SECTION: Diatom sample preparation
5. If samples are to be analyzed at a later date, samples should be stored in the fridge (max 48 hours), or flash frozen in   then stored at -80 °C .

SECTION: Flow cytometry analysis
6. Measure the background fluorescence of unstained cells using a flow cytometer. This protocol was developed using a CytoFlex LX.
 

Fluorescence is measured using 488 nm excitation and 525/40 nm detection.
Measure the background fluorescence of at least 200 cells but ideally 2000 or more. 
SECTION: Flow cytometry analysis
7. Add the BODIPY stain in a 1 % (v/v)  to final concentration of approx  0.002 mg/mL . For example add 2 µL to 200 µL  of microalgae sample, or 2.2 µL  to a fixed microalgae sample (200 µL sample with 20 µL  . Remember to take into account volume removed due to flow cytometry analysis.

SECTION: Preparation of BODIPY stock solution
1. Add  2 mg of   in powder form to 1 mL of   to create the stock solution of 2 mg/mL .

Store this in dark glass at -20 °C  to prevent degradation of the fluorescent dye.

SECTION: Flow cytometry analysis
8. Re-read the sample on the flow cytometer using the same laser parameters. Record the median fluorescence on the x channel for at least 200 cells

SECTION: Flow cytometry analysis
9. Calculate the change in median fluorescence between the stained and unstained sample. This is indicative of the level of neutral lipids present in the cell
SECTION: Flow cytometry analysis
10. If comparing between taxa of different sizes, size correction may be advisable. This may be done by dividing the change in median fluorescence by the equivalent spherical size, approximated from forward scatter which is measured in tandem during the flow cytometry analysis.
TITLE: Zeiss AxioImager Multi Channel 20X Image Capture
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the capture of 20X multi-channel images using human and mouse tissue.</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: Cell dissociation of fresh human lung tissue for single-cell RNA-seq
AUTHORS: Ilias Angelidis, Maximilian Strunz, Herbert Schiller

[STEPS]
?. Mince the tissue into small pieces using a pair of scissors. Transfer tissue chunks in a 50mL Falcon tube containing  of ice cold PBS. This step is intended to wash away any remaining blood off the tissue.
30 ml
?. Transfer lung tissue in a petri dish and cut the required amount for your experiment (2x2x1)cm.
?. Remove PBS by passing the minced tissue through a 40µm strainer and keeping the tissue pieces on top.
?. Transfer the tissue into a new 50mL Falcon tube containing  of  Enzyme Mix (should suffice for 2x2x1 cm of tissue).  ABC1EnzymeCat. NumberFinal Concentration2Dispase354235 (Corning)50 caseinolytic units/ml3CollagenaseC1-28 (Merck)0,6 mg/ml4Elastase20931 (Serva)0,02 mg/ml5DNase79254 (QIAGEN)17 units/ml
8 ml
ABC1EnzymeCat. NumberFinal Concentration2Dispase354235 (Corning)50 caseinolytic units/ml3CollagenaseC1-28 (Merck)0,6 mg/ml4Elastase20931 (Serva)0,02 mg/ml5DNase79254 (QIAGEN)17 units/ml
?. Incubate for: at with constant shaking at 750rpm.
?. After enzymatic digestion add of ice cold Inactivation Buffer. Mix well using a 10mL serological pipette and pass the cell suspension from the digested tissue through a 70μm strainer into a 15mL Falcon tube (keep flow-through).  ABC1ReagentCat. NumberFinal Concentration2PBS10010-015 (gibco)1X3FBSS 0615 (Merck)10%
7 ml
ABC1ReagentCat. NumberFinal Concentration2PBS10010-015 (gibco)1X3FBSS 0615 (Merck)10%
?. Centrifuge the cell isolate at 300g forat .
?. Resuspend cell pellet in of  RBC Lysis Buffer  at RT for to remove remaining red blood cells.
2 ml
?. Add of ice cold Inactivation Buffer to inhibit the activity of the RBC Lysis Buffer.
10 ml
?. Centrifuge the cell isolate at 300g for at .
?. Resuspend in of ice cold Inactivation Buffer and count the cells.
1 ml
?. Proceed with preferred scRNA-seq platform using the appropriate number of cells.
?. Assess cell viability using Trypan Blue staining. (cell viablity must be around 85%-95% in order to yield high quality single cell libraries)
TITLE: Sending samples to the NCI Frederick EM Core
AUTHORS: Bryce Killingsworth

[DESCRIPTION]
Protocol on how to ship EV sample to the NCI Frederick core.

[STEPS]
SECTION: Sending samples
1. If you are beginning a new project, you must put in a service request, select a CAN to draw funds from, and get approal from an AO, using the "New Request" button on the NCI Frederick EM Core's website: https://ncifrederick.cancer.gov/services/accessioning/services/labservices/area/5

Pg 1: information about the request: fill out scope of work and number of samples
Pg2: select CCR investigator and tag emails of anyone you want to add as followers
Pg3: Daily Cruz-Abreu is the Fiscal Authority. Funding source is the current fiscal year Project ID number located on the “General Info and Links” tab of the lab OneNote page.
SECTION: Sending samples
2. Ensure that Ziqui Wang or Kunio Nagashima at NCI Frederick is expecting your package delivery, and you have had communication about what type of images you expect.
SECTION: Sending samples
4. Thaw the EV samples you intend to have imaged.
SECTION: Sending samples
5. Transfer at least 50 µL of each of your sample to 0.5 mL tubes for shipment. It is possible as little as 35 µL could be imaged but any significant volume differences need to be discussed with the NCI core staff. Previously we have sent 100 µL per sample.
SECTION: Sending samples
6. Using a tube rack, other plastic supports, and tape, position the tubes in a plastic bag so that they are secure.
SECTION: Sending samples
7. Place the tubes in a sytrofoam container with ice from the ice machine, not dry ice. Use a paper towel as a barrier between the ice and the samples so they don't partially freeze.
SECTION: Sending samples
8. Create an item list using this template and print a copy to be shipped inside the box.

  

SECTION: Sending samples
9. Tape up the box.
SECTION: Sending samples
10. Affix a To and From label on the top of the box: 

TO: Ziqiu Wang
 Electron Microscope Facility NCI-Frederick
 EML at the ATRF
 8560 Progress Drive 
Wing D, Room D-2025 

FROM: Your Name
Jones Lab
Laboratory of Pathology
10 Center Dr
Bld 10, Rm B1B53
Bethesda MD,20892
SECTION: Sending samples
3. The Frederick Courier no longer accepts requests via email or phone. You must use the Shipping Wizard to arrange pickup.

https://ncifrederick.cancer.gov/cad/shippingwizard

NCI Frederick Courier: 301-846-1175 (contact number only, do not call for pickup)

Core Address:

TO: Ziqiu Wang
Electron Microscope Facility NCI-Frederick
EML at the ATRF
8560 Progress Drive 
Wing D, Room D-2025 
301-846-6064 

SECTION: Sending samples
11. You can wait for the courrier to knock on the lab door, or place the package with a note for the courrier outside the lab in the hallway.
SECTION: Sending samples
12. Email Ziqui and Kunio to let them know the shipment is on the way.

nagashimak@mail.nih.gov

ziqiu.wang@nih.gov
TITLE: No Title
AUTHORS: 

[DESCRIPTION]


https://wickie.hlrs.de/platforms/index.php/Cray_XC40

[STEPS]
SECTION: Getting access to Hazel Hen
?. 
SECTION: Test access and set up a workspace
?. 
SECTION: Set up ssh
?. 
SECTION: Get code via ssh+git
?. 
SECTION: Prepare and build
?. 
SECTION: Running jobs
?.
TITLE: Elisa
AUTHORS: Alex Andre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Titration of antibodies in the serum of patients.</div></div>

[STEPS]
?. [coating]
add ab at
1 Micromolar (µM)
?. [coating]
wash and keep at
?. [Blocking]
add 100ul BSA
?. [Blocking]
incubate
?. [Adding of sample]
add the serum in the plate
?. [Adding of sample]
incubate
?. [Adding of sample]
Wash
?. [Detection]
Add Ab at
10 Micromolar (µM)
?. [Detection]
incubate
TITLE: Artificial Cerebrospinal Fluid VII (ACSF.VII)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare Artificial Cerebrospinal Fluid VII (ACSF.VII). ACSF.VII is used for applications including fresh human brain tissue transportation and as a bathing solution during fresh human brain slicing.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]