eamag/protocols-with-steps huggingface dataset:
TITLE: Preparation of mouse tissue homogenates for RT-QuIC assay
AUTHORS: arpine.sokratian, andrew.west

[DESCRIPTION]
This protocol is designed for a standardized and efficient procedure to homogenize mouse tissue, conducive for RT-QuIC analysis. The process involves treating samples with PBS mixed with Triton-X100, followed by homogenization using a prob-tip sonicator. Centrifugation is then employed to remove detergent-insoluble fraction, optimizing the tissue for subsequent 
analysis. The use of Triton-X100, a mild detergent, ensures the release of alpha-synuclein aggregates, enhancing signal specificity without compromising structural integrity. The protocol's significance lies in addressing the critical need for a reliable method in preparing mouse tissue for RT-QuIC analysis, with the distinct signal generated in assays validating its efficacy. 

[BEFORE_START]
The tissues need to be perfused with fresh PBS and flash-frozen in liquid nitrogen

[STEPS]
1. Measure the weight of each eppendorf tube using analytical scale and label the tube with assigned number
  
   
2. Cut a tissue slice (around 100-200 mg) with a disposable blade on a plastic plate lid wrapped in a foil (all on ice), transfer to the Eppendorf protein low-binding tube (with known weight)
 

3. Measure the weight of the tissue piece in a tube using analytical scales 
Subtract weight of the tube to obtain the final weight of the brain slice (indicate the weight in the table)
4. Add 20 vol weight/volume of PBS buffer containing 1%Tx100 and protease/phosphatase inhibitors (  ,  (for example, 100 mg – 5 mL)  - transfer to 15 mL falcon tube;

5. Sonicate tissue samples for 1 min, 5 sec ON and 15 sec OFF (on ice);
Use 0.16 inch microtip for homogenization
 

6. Spin down sonicated samples at 10.000 x g, 4 °C, 10 min

7. Transfer supernatant into a new 15 mL falcon tube;

8. Aliquot the samples in lobind eppendorf   tubes and store at -70 °C
TITLE: Site Directed Mutagenesis 2016
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Site--directed Mutagenesis</div></div>

[STEPS]
?. [DpnI Digest:]
Transfer PCR to Epp tube.
?. [DpnI Digest:]
Remove 2 L from PCR.
?. [DpnI Digest:]
Add 2 L DpnI enzyme to remaining 48L.
?. [DpnI Digest:]
Incubate 1+ hrs 37ºC (1st digestion).
?. [DpnI Digest:]
Add another 2 L DpnI enzyme to remaining 48 L (2nd digestion).
?. [DpnI Digest:]
Use PCR purification kit to purify the 2nd digestion product.
After PCR purification, you will lose around 20% of your product. I always try to concentrate my 2nd digestion PCR product to a volume of around 40 L before I do the transformation in order to keep the same concentration between the undigested product and the digested product.
?. [DpnI Digest:]
Store at -20  ºC.
You can go ahead to do the transformation using the undigested PCR product and the 2nd digestion product. If you don't get quite good result after the transformation, such as no significant difference between the undigested one and the 2nd digestion one, you can do the 3rd digestion. But when you do the 3rd digestion, you need to add restriction enzyme buffer 4.
?. [Transformation:]
Make TOP10  electrocompetent cells as usual.
?. [Transformation:]
Transform 1L DpnI-digested plasmid and 1L non-DpnI plasmid as control.
?. [Transformation:]
Plate 1/10, 9/10 on LB + antibiotic plates.
?. [Transformation:]
Should see many more colonies on the -DpnI plate if the digest of the parental unmutated plasmid worked well.
?. [Transformation:]
Streak a minimum of 6.
?. [Transformation:]
Sequence 6.
?. [Transformation:]
Check thatthe desired mutation is present .the primer region did not get duplicated. Sometimes you will see that somehow the 60-mer of the primer is present in two or more tandem repeats.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Reliable clinical tests that are capable of measuring resistance are important tools for rehabilitation. An alternative that has increased popularity is the use of elastic tubes, which stand out for being easy to handle, low cost, practicality, and feasibility of use. To analyze the test-retest reliability of the muscle fatigue resistance test (FRT) with elastic tubes. A total of 116 healthy young males, age between 18 and 30 years old, participated in the study. Participants performed three pre-test stages: orientation, load presentation, and familiarization with equipment lasting two weeks. Subsequently, they performed the FRT on two occasions (test and retest), with an interval of seven days. The reliability analyzes were performed using the intraclass correlation coefficient (ICC) with 95% confidence interval and typical measurement error (TME), also expressed as coefficient of variation (CV%). The findings regarding the reliability of the test showed satisfactory values (time: ICC= 0.66; 95%CI [0.50; 0.76]; CV(%)= 9.34; repetition: ICC= 0.61; 95%CI [0.46; 0.73], CV(%)= 13.66; rhythm: ICC= 0.52; 95%CI [0.35; 0.67], CV(%)= 10.29). From the findings presented, it is concluded that the clinical test proposed with elastic tubes demonstrates evidence of acceptable values.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Standard method for microCT-based additive manufacturing quality control 5: witness specimen analysis
AUTHORS: Anton du Plessis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">MicroCT is a well known technique and is often used for non-destructive analysis of parts produced by additive manufacturing. This is especially useful to inspect the parts for potential defects such as excessive porosity or layered defects due to imperfect powder levelling in individual layers. While various online monitoring methods are under development, there is currently not yet any accepted method to identify such defects during the building process. Post-production microCT of the entire part allows for the identification of such errors, but its limitation is the resolution which depends on part size. Hence a large part may have small but important porosities which may be missed by microCT. This can be overcome by using a witness specimen built in the same process, and analysing this smaller sample at a high resolution. The porosity information from the smaller part can be used to evaluate the acceptability of the entire build. The workflow presented here serves to standardize this process: with a simple process, a standard geometry witness specimen and hence standardized scan and analysis procedures. A layered stop-start flaw is shown in the witness specimen to demonstrate the ability to detect this type of flaw.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Viral Informatics Resource for Metagenome Exploration (VIROME) is a bioinformatics pipeline that classifies viral metagenome sequences after searching against annotated reference sequence databases (i.e., UniRef, SEED, ACLAME, COG, GO, KEGG, and PhageSEED) and a custom environmental database, Metagenomes Online (MgOl). VIROME’s compare tool allows users to compare any number of metagenomes to one another by generating a Biological Observation Matrix (biom) file that contain counts of occurrences of custom observations (comparison metrics), and a mapping text file that provides per-sample metadata (library IDs and descriptions).</p>
<p> Quantitative Insights Into Microbial Ecology (QIIME) is an open-source software pipeline that allows users to interpret raw sequencing data and create graphical displays to interact with the data. QIIME is modular, providing users with the flexibility to select and integrate various applications. </p>
<p>While QIIME has many capabilities and online tutorials, this SOP is limited to the steps a user will take to use VIROME’s compare tool output (biom and mapping files) to explore composition (what is in a sample), alpha-diversity (diversity within a sample), and beta-diversity (comparison of diversity between samples). This SOP generally follows guidance in online tutorials but with modifications unique to the VIROME Comparinator output analysis. </p>

[BEFORE_START]
<p>Make sure you have QIIME installed or are using the QIIME virtual machine.<br /><br />Tutorials are available online at:</p>
<p><a href="http://qiime.org/tutorials/index.html" target="_blank">http://qiime.org/tutorials/index.html</a></p>
<p>This SOP was drafted based on tutorials available at the time of writing, including the 454 Overview Tutorial:</p>
<p><a href="http://qiime.org/tutorials/tutorial.html" target="_blank">http://qiime.org/tutorials/tutorial.html</a> and</p>
<p>Illumina Overview Tutorial:</p>
<p><a href="http://nbviewer.ipython.org/github/biocore/qiime/blob/1.9.1/examples/ipynb/illumina_overview_tutorial.ipynb" target="_blank">http://nbviewer.ipython.org/github/biocore/qiime/blob/1.9.1/examples/ipynb/illumina_overview_tutorial.ipynb</a></p>

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?. 
?. 
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?. 
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?. 
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?. 
?.
TITLE: Shenfu Injection as an add-on treatment to improve survival rate of patients after cardiopulmonary resuscitation: a meta-analysis (protocol)
AUTHORS: Shuo Zhang, Xiaobei Si, Xiao-Han Fan, Lin-Yu Huo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objective </span><span>To evaluate the improvement of survival rate for patients after cardiopulmonary resuscitation (CPR) by Shenfu Injection. </span><span style = "font-weight:bold;">Method </span><span>Electronic databases including Medline, Embase, Web of Science, Cochrane Central Register of Controlled Trial, Wanfang Database, and VIP Database were searched with the search terms of "Shenfu Injection”, “cardiopulmonary resuscitation”, “randomized controlled trials” and their synonyms. A funnel plot was adopted to evaluate publication bias. The meta-analysis was performed using a fixed effects model in case of low heterogeneity and a randomized effects model in case of high heterogeneity. </span><span style = "font-weight:bold;">Conclusion </span><span>Add on treatment of Shenfu Injection may improve the survival rate of the patients after CPR. </span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a multiplex PCR-SSP for amplification of the follow SNPs of CR1 gene:</p>
<p>rs3737002;</p>
<p>rs11118131; </p>
<p>rs11118167; </p>
<p>rs17047660</p>

[BEFORE_START]
<p>1- Wear clean gloves;<br />2- Clean pipettes and stand with hypochlorite and 70% alcohol;<br />3- Defreeze DNA samples and reagents (exception: Taq polymerase);<br />4- Gently mix the DNA samples and pass the reagents briefly on the vortex.<br />5- Centrifuge the DNA samples and reagents with a spin;<br />6- Keep reagents on ice;<br />7- Leave microtubes ready for preparation of the mix, keep the same ones identified and on ice.<br />8- Leave application plates properly identified.</p>

[STEPS]
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?. 
?. 
?. 
?. 
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?.
TITLE: Keto Max Science Canada - Reviews, Benefits, Side Effects
AUTHORS: ketomaxciencecanadas  

[DESCRIPTION]
Keto Max Science Canada - Reviews, Benefits, Side Effects


[STEPS]
1. Keto Max Science Canada - Reviews, Benefits, Side Effects
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It moves seriously when it's fueled with unalloyed heat. Weight loss supplements can help you lose redundant water weight. While this may make you feel slimmer and further energetic, it'll not help with your water weight. This form doesn't do that. It doesn't contain any fat. To get the smallest Keto Max Science price of the period, snap any image on this runner! 
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The ketones in the equation tell your body that it's a great time to enter ketosis. Open your fat stores and eat them with smoldering warmth. This form will help you get relieve of redundant fat, similar as your stomach doggy, protuberance, and back fat. 
What's Keto Max Science Weight Loss Supplement? 
These are the data. Why is the internet buzzing about this product? You presumably saw some online elevations for Keto Max Science Pills and wondered if they were really worth it. We're assuming that this item is being liked by people who have entered positive reviews. We have n’t seen a keto diet lozenge explode like this for quite some time. You can also get on board the keto style to see if it helps you! 
According to Keto Max Science weight loss capsules reviews, guests feel more energetic than ever. Some indeed claim they no longer drink coffee in the morning. According to different checks, this product encouraged people to eat further fat than they ever imagined. However, you can let your body do all the work, If you're too busy to suppose about losing weight. 

Official Website:- http://softtechfreak.com/health/keto-max-science-canada-reviews-price-benefits-side-effects/
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Data sources: Pubmed, Cochrane, Web of Science, Science Direct, Scopus, and Ovid MD. Searched until 30th April 2017. (chlorhexidine) AND (bacteremia OR bacteraemia) AND (extraction OR removal) were used as key words in a free-text search. Meeting Abstracts published were searched. References of each article were reviewed. We only included randomized controlled clinical trials. There were no restrictions regarding language or date of publication. The outcome measure was set on the incidence of the bacteremia measure within the first ten minutes post-extraction. Two reviewers independently undertook the risk of bias assessment and data extraction. A fixed-effect inverse variance weighted meta-analysis was conducted.</p>

[STEPS]
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describe the steps to perform <em>Chlamydomonas reinhardtii</em> fluorescence microscopy.</p>

[BEFORE_START]
<ul>
<li>Culture cells in liquid media</li>
</ul>

[GUIDELINES]
<p>Cells should be cultured to a late log phase to increase cell number for microscopy.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks
AUTHORS: Sang Hyo Kim, Caleb  Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier  D. Monzón

[DESCRIPTION]
This protocol details a novel method to isolate individual ticks and manipulate their environment. We successfully used this method to investigate how humidity affects survival and host-seeking (questing) behavior of three species of ticks: the lone star tick (Amblyomma americanum), American dog tick (Dermacentor variabilis), and black-legged tick (Ixodes scapularis). We placed 72 adult females of each species into individual plastic tubes and separated them into three experimental relative humidity (RH) treatments representing distinct climates: 32% RH, 58% RH, and 84% RH. For 30 days we assessed the survival and questing behavior of each tick.

[BEFORE_START]
We acquired adult female ticks from the Oklahoma State University Tick Rearing Facility and experimented with one temperature range and three relative humidities. This protocol can be modified for different species, life stages, temperatures, humidities, and other small organisms. 

[GUIDELINES]
Questing qualifications: 
The individual is not walking but it is still with its front legs extended       

Death qualifications: 
If any tick appears dead, lightly blow on it since ticks respond to carbon dioxide exhaled by potential hosts
If the tick does not move at all in 2 minutes, it should be counted as dead and placed in 70% ethanol

[STEPS]
SECTION: Set up
1. Place a single tick with one wooden skewer in each tube and seal with a cap, labelling each tube with an individual identifier  
  
SECTION: Set up
2. Place six tubes in each airtight container along with a humidity pack, labelling each container
   

SECTION: Set up
2.1. Confirm the humidity in one container of each RH level with the data logger 

SECTION: Set up
3. Program the climate chamber
 

SECTION: Set up
3.1. To cycle between 20 °C to 30 °C, the temperature increments should be as follows: 
3:00  - 25 °C 
6:00  - 27.5 °C 
9:00  - 30 °C 
12:00  - 27.5 °C 
15:00  - 25 °C 
18:00  - 22.5 °C 
21:00  - 20 °C 
24:00  - 22.5 °C 


SECTION: Set up
3.2. To create a 12:12 light:dark photoperiod, lighting increments should be as follows:
9:00 - Lights on
21:00 - Lights off
SECTION: Data collection
4. Place all of the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program
SECTION: Data collection
5. Each day thereafter, during the 9:00 to 12:00 or 30 °C increment, assess each tick for survivorship and questing behavior (see guidelines for qualifications of survivorship and questing)
 

 


SECTION: Data collection
5.1. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers has occurred
SECTION: Data collection
6. Repeat steps 4 and 5 for 30 days or until all ticks have died
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a controlled, randomized, and cross-over investigation with 40-week duration. The participants were male young adults with previous experience with RT for testing the effect of long-term RT with different volumes on HRV. This study was conducted in healthy participants in order to investigate the effects of resistance training on HRV without pathological influences. They underwent to a 40-week intervention divided in three RT segments (8 weeks each) and two washouts (8 weeks each) according Figure 1. The intervention order was randomized and counterbalanced. Thus, participants performed 1, 3 or 5 sets of the same exercises with equalized intensity (loading zones) and rest (Table 1) for 8 weeks following 8 weeks of washout between each experimental condition (1 vs. 3 vs. 5 sets).</p>
<p>10 repetition maximum (10-RM) test was performed for defining training intensity that was adjusted weekly (increase/decrease of 2-5% and 5-10% for upper and lower limbs, respectively) as recommended by Ribeiro et al. (2016). In case the participant performed more than 10 repetitions in two sessions, load (kg) was increased in the next week, whereas whether the participant was not able to perform 10-RM the load was decreased in the next week.</p>
<p>            HRV was assessed by cardiac monitoring 72h before (pre) and after (post) each experimental RT condition (1 vs. 3 vs. 5 sets). Moreover, the participants were instructed to not performing any physical exercise 48h previously HRV baseline evaluation.</p>

[GUIDELINES]
<p>American College of Sports Medicine</p>

[STEPS]
TITLE: Chlorophyll Extraction and Spectral Analysis with Spectrometer Calibration
AUTHORS: Victor Rodriguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to be able to extract  and analyze the concentration of chlorophyll within a sample of a given plant. The procedures of this protocol require using a spectroscope  to determine the approximate level a chlorophyll  within a given sample.</div></div>

[STEPS]
?. [Extraction of Chlorophyll ]
Weigh out  of sample subject and add it to a pestle.
0.25 g
?. [Extraction of Chlorophyll ]
Weigh out  of  and add it to a pestle.
0.13 g
?. [Extraction of Chlorophyll ]
Add  of to the pestle.
1 ml
?. [Extraction of Chlorophyll ]
Grind the entire mixture until the sample is consistent and theis completely dissolved into the sample paste.
?. [Extraction of Chlorophyll ]
Add the paste into a 40 ml beaker and wash out the pestle with   of  and empty it into the beaker.
1 ml
?. [Extraction of Chlorophyll ]
Add  of to the 40 ml beaker and mix the sample thoroughly, ensuring to press and remix the sample several times.
13 ml
?. [Extraction of Chlorophyll ]
Let the sample beaker stand for 15 minutes.
?. [Extraction of Chlorophyll ]
Filter the sample into a 20 ml test tube using a 20 micron retention filter paper.
?. [Extraction of Chlorophyll ]
Let the sample stand for a further 10 minutes.
?. [Sample Analyzation ]
Add  of the sample to two PCR tubes and label them according to your needed labeling system. the two tubes will serve as double tests for verification.
200 µl
?. [Sample Analyzation ]
Centrifuge the two samples to 1200 g to settle out any particulates for 1 minute.
?. [Sample Analyzation ]
From each PCR tube, transfer  of the sample from the top to another PCR Tube.
100 µl
?. [Sample Analyzation ]
Place each tube in the spectrometer.
?. [Sample Analyzation ]
Take to log base 10 of the absorbance percentage of the wavelength 647 nm and 664.5 nm and denote them as A647 and A664.
?. [Sample Analyzation ]
To calculate the concentration...CHLA=20.47A647 - 4.73A664CHLB= 12.63A664 - 2.52A647CHLTotal= CHLA + CHLBDivide each concentration by 1000, multiply by the acetone used, then divide by the sample mass to get the mg/g of sample
?. [Sample Analyzation ]
Confirm readings match to within an acceptable margin of error.
?. [Sample Analyzation ]
Add   of to a PCR tube and label it "base"
100 µl
?. [Sample Analyzation ]
Use the "base" PCR tube to calibrate the spectrometer.
TITLE: Content of the therapeutic protocol
AUTHORS: Mohammad Tahan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Animal-Assisted Therapy </span></div></div>

[STEPS]
?. Introduction, briefing, primary assessing and introduction to the course
?. Giving an introduction about animals and their specific characteristics
?. Taking children to the place that animals were kept
?. Spending time in the place that animals were kept, talking about them and how to communicate with them
?. Convincing children to come closer and touch and pet them
?. Playing with animal and feeding them
?. Participation in activities like washing and feeding the animals
?. Summarizing, giving feedback, and post-test
TITLE: Intracellular Staining of PUMA in Primary PBMC Lymphocytes
AUTHORS: Dennis Juarez, dfruman

[DESCRIPTION]
Flow cytometric assessment of Intracellular PUMA levels is useful when wanting to assess PBMC subsets but are limited by sample as with primary patient samples. Here I describe a protocol for intracellular PUMA staining validated with PUMA knockdown and induction with PUMA inducing treatments.

[STEPS]
SECTION: Staining
1. Treat at least 1 million PBMCs per sample, including flow cytometry controls (unstained, single stain controls, and FMO controls)
SECTION: Staining
2. Harvest cells at 20 hours and wash with PBS.
SECTION: Staining
3. Stain cells with anti-CD3, anti-CD4 and anti-CD19 for 20 minutes at 4 degrees in the dark. Create unstained, single stain controls, and FMO controls.
SECTION: Staining
4. After staining, wash with PBS and resuspend 1 million cells in 100uL of PBS-diluted 2% formaldehyde for 10 minutes at RT.
SECTION: Staining
5. Add 1mL of PBS and spin down the cells at 800xg for 5 minutes.
SECTION: Staining
6. Wash two times with 1x intracellular staining buffer (see materials).
SECTION: Staining
7. Stain for intracellular PUMA in 1x intracellular staining buffer: 50ul staining volume per sample with 1:50 final dilution Puma (Cell signaling D30C10) Rabbit mAb
SECTION: Staining
8. Stain at 4 degrees for 1 hour in the dark.
SECTION: Staining
9. Spin down and wash two times with 1x intracellular buffer.
SECTION: Staining
10. Prepare the secondary Invitrogen’s anti-Rabbit IgG 647 #A-21244  at Final Dilution 1:500 in 1x staining buffer. Be sure to stain the PUMA FMO with the secondary antibody.
SECTION: Staining
11. Stain for 1 hr in the dark at RT.
SECTION: Staining
12. Wash twice with 1x intracellular buffer and once with PBS. Resuspend in 100-150uL PBS and samples are ready to run.
SECTION: Validation
13. Protocol used in PUMA knockdown cell line validated by western blot.
   

SECTION: Validation
14.   

SECTION: Validation
15.