eamag/protocols-with-steps huggingface dataset:
TITLE: Keto Start ACV Gummies Reviews &amp; Shark Tank, Price, Benefits
AUTHORS: ketostartgummiesreviews  

[DESCRIPTION]
Keto Start ACV Gummies Reviews & Shark Tank, Price, Benefits

[STEPS]
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1.
TITLE: Physical property-Stability and pH (TBE and Borax Agarose electrophoresis buffer)
AUTHORS: Stephane  Fadanka, Nadine   Mowoh

[DESCRIPTION]
The pH check is important because changes in the pH might affect mobility of DNA in the agarose gel hence PCR result alteration and misinterpretation.
The physical stability of the electrophoresis buffer is important because if the powder absorbs moisture from air overtime, they might not be very efficient in allowing migration of DNA samples in the gel which could affect the PCR results.
This protocol describes how to access the physical property of TBE and Borax electrophoresis buffers.

[BEFORE_START]
Ensure all preliminary steps involved in the process are performed (e.g initially weighing and drying buffer powders)

[GUIDELINES]
Quality control tests are important to ensure the reagents are functioning properly. In this procedure we describe how to access the physical stability and pH of Agarose electrophoresis buffer as this might affect the migration of DNA sample in an Agarose gel electrophoresis.

[STEPS]
SECTION: Confirming Stability and pH
1. 
 

To confirm the weight of the powder buffer sachet:

Weigh the individual powder components that constitute the buffer ( the individual powders could be pre-dried if necessary in an incubator with silica gel beads at 37 °C)
Put them together in a beaker and mix.
Pour the powder mix in a zip lock sachet and store in an air tight container for 1 to 3 days.
After, open the sachets and pour the powder into a weighing boat and measure the weight of the dried powder.
The weight should be approximately close to or equal to the weight of the powder before the 3 days storage.

To confirm the pH of the buffer solution:

Pour the powder buffer into an appropriate size beaker and add the appropriate amount of distilled water as indicated on the powder sachet (e.g 1 sachet in 1L water).
Mix until a clear solution is obtained
Use a pH meter to check the pH of the solution which should be between 8.3 and 8.5.
TITLE: Set of quality indicators for physiotherapy care process of patients with Whiplash-Associated Disorders (WAD)
AUTHORS: Rob A B Oostendorp

[DESCRIPTION]
Quality indicators for physiotherapy care process of patients with Whiplash-Associated Disorders (WAD):
. steps of clinical reasoning
. number of indicators
. type of indicator
. item measured
. indicator
. level of evidence
. performance target

[STEPS]
TITLE: Recordings of cervical vagus nerve activity
AUTHORS: Vitaly Napadow, Ilknur Ay, Catherine Morello

[DESCRIPTION]
The goal of this work is to evaluate laterality of vagus nerve efference evoked by unilateral auricular branch of the vagus nerve (ABVN) stimulation by performing bilateral recordings of cervical vagus nerve activity (CVNA).

[STEPS]
3. CVNA was recorded using hook electrodes from the right and/or left cervical vagus nerve in response to left auricular vagus nerve stimulation.
SECTION: fromNewSection
1. Ten-to-12 week old male and female Wistar rats ranging in weight from 232-400g were anesthesized.
SECTION: fromNewSection
4. ABVN stimulation protocol was as follows: three 0.5mA trains of stimulation (1Hz, 0.5ms) repeated at every 5min followed by three 1mA trains of stimulation (1Hz, 0.5ms) repeated at every 5 min. Parameters for ABVN stimulation included a series of 5 stimulus pulse trains, with each pulse consisting of a 0.5 mA current intensity, 0.5 ms pulse width, and delivered at a frequency of 20Hz. 
SECTION: fromNewSection
2. Stimulation was delivered to cymba and cavum conchae using a Grass S48 stimulator in conjunction with a Grass stimulus isolation unit and constant current unit stimulator.
TITLE: LR Clonase
AUTHORS: Invitrogen

[STEPS]
?. Add the following components to a 1.5-mL microcentrifuge tube at room temperature and mix: • 1–7 μL entry clone (50–150 ng) • 1 μL destination vector (150 ng/μL) • TE bufferǰ pH 8.0, to 8 μL
?. Thaw on ice the LR Clonase™ II enzyme mix for about 2 minutes. Vortex the LR Clonase™ II enzyme mix brefl¢ twice (2 seconds each time).
?. To each sample (step 1), add 2 μL of LR Clonase™ II enzyme mix to the reaction and mix well by vortexing brefl¢ twice. Microcentrifuge brefl¢ǯ
?. Return LR Clonase™ II enzyme mix to –20°C or –80°C storage.
?. Incubate reactions at 25°C for 1 hour. (overnight)
?. Add 1 μL of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly Incubate samples at 37°C for 10 minutes. Transform
?. Transform 1 μL of each LR reaction into 50 μL of One Shot™ OmniMAX™ 2 T1 Phage-Resistant Cells (Cat. no. C8540-03) (5 alpha e. coli). Incubate on ice for 30 minutes. Follow electroboration protocol.
?. 
?. T1 Phage-Resistant Cells as described above. Plate 20 μL and 100 μL on LB plates containing 100 μg/mL ampicillin. Expected results An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">This protocol has been optimized for </span><a href="&quot;https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system.html?c" target="_blank" rel="noopener noreferrer"><span style="font-weight: 400;">Thermo Fisher’s Neon Electroporation System</span></a><span style="font-weight: 400;"> and is based on the report </span><a href="https://www.biorxiv.org/content/early/2017/12/07/183418" target="_blank" rel="noopener noreferrer"><span style="font-weight: 400;">Reprogramming human T cell function and specificity with non-viral genome targeting</span></a><span style="font-weight: 400;">. We have used </span><a href="https://www.thermofisher.com/order/catalog/product/A36497" target="_blank" rel="noopener noreferrer"><span style="font-weight: 400;">TrueCut™ Cas9 Protein v2</span></a><span style="font-weight: 400;"> and reached very comparable KO efficiencies to those previously shown by others.</span></p>

[GUIDELINES]
<p><span style="font-weight: 400;">To reduce variation per electroporation sample and to account for practical issues of electroporation (e.g. sparks), we have also found that a 24-well plate setup where we seeded cells from three independent electroporation reactions (within 10-ul tip) into a single well with 500 ul to 1 ml culture media in it had been the most feasible approach. This setup is especially helpful during initial optimization experiments where the goal is to screen many conditions. Pooling three independent reactions into a single sample provides a virtual buffer against variation across electroporation samples and yields enough treated cells for common assaying approaches, such as Western Blotting. We always recommend optimizing or screening using 10-ul tip setup and then scaling the reactions up to 100-ul once the optimal settings are known.</span></p>

[STEPS]
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TITLE: The TARGET System: Rapid Identification of Direct Targets of Transcription Factors by Gene Regulation in Plant Cells
AUTHORS: Matthew D. Brooks, Kelsey M. Reed, Gabriel Krouk, Gloria M. Coruzzi, Bastiaan O. R. Bargmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, makes this plant cell-based approach a valuable tool for a higher through-put approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.</div></div>

[STEPS]
TITLE: AAV DNA library generation
AUTHORS: Sripriya Ravindra Kumar, Timothy F. Shay, Xinhong Chen, David Brown, Tatyana Dobreva, Qin Huang, Xiaozhe Ding, Yicheng Luo, Pétur H. Einarsson, Alon Greenbaum, Min J. Jang, Benjamin E. Deverman, Viviana Gradinaru

[DESCRIPTION]
This protocol describes how to generate a DNA library of AAV capsid variants diversified by insertion of a randomized sequence encoding 7 amino acids between AA588 and AA589 of AAV9.

[STEPS]
SECTION: Generation of Library Fragments
1. Design primers for the randomized insertion. 
 

SECTION: Generation of Library Fragments
2. Generate the AAV capsid library fragments by PCR using the AAV9 cap gene as template with   and forward and reverse primers.

 


SECTION: Library Assembly
5. Linearize the rAAV-ΔCap-in-cis-Lox plasmid by restriction digest with AgeI and XbaI
SECTION: Generation of Library Fragments
3. Run PCR products on a 1% agarose gel.
SECTION: Generation of Library Fragments
4. Purify the 480 bp band with   
 


SECTION: Library Assembly
6. Insert the amplified library fragments into the linearized vector in a 1:2 molar ratio using   

SECTION: Library Purification
7. Treat with either   or   to degrade non-assembled DNA fragments remaining in the mixture.

SECTION: Library Purification
8. Purify assembled library with   

SECTION: Library Quality Validation
9. Transform 1 ng assembled library into   and check for colonies after overnight incubation at 37ºC on LB-agar plates containing carbenicillin.
SECTION: Library Quality Validation
10. Sequence the DNA library around the insertion site. A non-biased library should match the diversity of the NNK/NNM motif (N=25% each of A, T, G and C; K=50% each of G and T; M=50% each of A and C) with minor fluctuations.
SECTION: Library Quality Validation
11. To verify that the ITRs are intact, digest with   

SECTION: Library Quality Validation
12. Transfect   with 10 ng of library. Uniform expression of mNeonGreen should be observed across cells. Measuring the average yield per dish will inform scaling for full production.
TITLE: Immunohistochemistry of liver tissue sections
AUTHORS: Presha   Rajbhandari, Taruna  V. Neelakantan, Brent  R. Stockwell

[DESCRIPTION]
This protocol outlines the steps used to perform standard immunohistochemistry for antibody validation in frozen human liver tissue samples, performed at Molecular Pathology Core facility at Columbia University.

[STEPS]
1. Cryosection the frozen liver tissue at 5µm thickness and place it on charged slide

2. Air dry the sections for 3 minutes 180 min 

3. Fix the tissue sections in cold acetone for 15 minutes 15 min  

4. Air dry the sections at room temperature for 2 minutes 2 min  

5. Incubate the slides in 0.3% hydrogen peroxide in PBS for 5 minutes 5 minto block peroxidase activity

6. Wash slides with PBS 3 times, for 5 minutes each 5 min 

7. Block the tissue sections in 10% normal goat serum or 5%Horse serum with 0.1%BSA for 20 minutes 20 min 

8. Incubate the tissue sections with primary antibody diluted in DAKO antibody diluent at room temperature for 1.5-2 hours 90 min 120 min 

9. Wash the slides with PBS 3 times, for 5 minutes each  5 min 

10. Incubate the tissue sections with biotinylated secondary antibody diluted in PBS at room temperature for 30-45 minutes 30 min 45 min 

11. Wash the slides with PBS 3 times, for 5 minutes each  5 min 
12. Incubate the tissue sections with ABC (Avidin-Biotin complex) peroxidase solution at room temperature for 30 minutes  30 min 

13. Wash the slides with PBS 3 times, 5 minutes each  5 min 

14. Incubate the tissue sections with DAB (3,3’-diaminobenzidine) peroxidase substrate solution until desired color intensity is reached and immerse slides in distilled water
15. Counterstain with hematoxylin and rinse with distilled water
16. Dehydrate the sections using 95% ethanol followed by 100% ethanol
17. Clear with xylene and mount coverslip using mounting medium
TITLE: Test for admin publication
AUTHORS: Joanna Sendecka

[DESCRIPTION]
This protocol is created to test the process of publishing by admins and possible curation possibilities. We want to see possible notifications to admins and how the process works.

[GUIDELINES]
This protocol is created to test the process of publishing by admins and possible curation possibilities.

[STEPS]
SECTION: Section one and only
1. This is step one
SECTION: Section one and only
1.1. This is sub-step two
SECTION: Section one and only
1.2. This is sub-step two
SECTION: Section one and only
2. Step two
TITLE: Growth Curves of S. elongatus under salt stress and high carbon
AUTHORS: Akashdutta  

[DESCRIPTION]
When liquid media is inoculated with bacteria and the cell population is counted at intervals, it is possible to plot a typical bacterial growth curve that shows the increase in the number of cells over time. Such growth curves show four distinct phases of growth:1

Lag phase: There is slow growth or lack of growth due to the physiological adaptation of cells to culture conditions or the dilution of exoenzymes (due to initial low cell densities).
Log or exponential phase: Optimal growth rates are seen in this phase. Cell numbers double at discrete time intervals known as the mean generation time.
Stationary phase: During this phase, the growth (cell division) and death of cells occur at the same rate, resulting in the number of cells being constant. The reduced growth rate is usually due to a lack of nutrients and/or a buildup of toxic waste constituents.
Decline or death phase: Here, the death rate exceeds the growth rate, resulting in a net loss of viable cells.

This is one of the simplest methods used to analyze trends in growth because it uses a spectrophotometer to track changes in the optical density (OD) over time. In other words, as the number of cells in a sample increases, the transmission of light through the sample will decrease.2

Growth curves for certain freshwater cyanobacterial species are carried out under salt stress to account for sucrose production in the particular strain. This is because sucrose is naturally produced intracellularly in these strains to balance the osmotic pressure of a saline environment.3

This experiment was carried out in two iterations: one at 0.04% of carbon dioxide -  i.e, ambient carbon dioxide from the atmosphere - and the other with bicarbonate added to be equivalent to 0.6% of carbon dioxide. Find our results for these iterations here.


[STEPS]
1. Grow 100 mL of culture in a 250 mL flask in CoBG-11 medium.
2. To this add bicarbonate equivalent to 0.6% carbon dioxide., or 2 g/L of sodium bicarbonate in CoBG-11
3. 12 hours later, take 3 1 mL  samples from each flask in 2 microcentrifuge tubes
4. Use one set of samples to take the OD measurements 
5. Take the other set of samples and centrifuge at 4000 rpm, 7 min, 25 °C 
6. Take the supernatant in fresh microcentrifuge tubes and store it at -20 °C , for later analysis. Discard the pellets.

7. Take the readings every twelve hours until the OD730  value reaches 1.0
8. Add the bicarbonate at regular intervals of initially once every 24 hours, increasing the frequency if the culture turns a dull yellow.
TITLE: Genomic RNA extraction by TRIzol method by using Drosophila melanogaster  gut
AUTHORS: Mohamed Sabeelil  Islam

[DESCRIPTION]
Simple method followed for RNA extraction of Drosophila melanogaster larval(3rd instar) gut for CDNA

[GUIDELINES]
ware gloves while extraction 

[STEPS]
1. Dissection was used to isolate the larval gut of Drosophila 3rd Instar.
(around 10-15 gut was taken)
2. 
The dissected gut was taken in a 1.5ml ependorf tube which contained 200μL of TRIzol.

(For a total of 800μL or 0.8 mL of TRIzol needed for the extraction)
To avoid spurt, 200μL were taken.

3. Homogenize by using a micropistle

4. Add 600μL or 0.6mL of TRIzol followed up by adding 200μL or 0.2mL of Chloroform
(after adding vortex it) and keep it for 5 minutes incubation

5. Centrifuge at 12000x for 10minutes at 4ºC
6. Take supernatant into new tube and add equal amount of isopropanol 
7. Centrifuge at 12000x for 10minutes at 4ºC
8. discard the supernatant and retain the pellet 
8.1. wash the pellet by using 1ml of 75% cold ethanol (twice) 
(after adding ethanol vortex the solution and centrifuge at 7500x for 5 minutes)

9. Retain the pellet, add RNase free water and check the purity
TITLE: Animal Room (Covid-19 Shutdown Re-Entry, Srivastava Lab)
AUTHORS: Srivastava Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Disclaimer: </span><span>The SOP presented here has been designed by the Srivastava Lab at Harvard University and is being shared outside of Harvard for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
?. Wash hands before entering the animal room.
If you choose to wear gloves, be cognizant not to touch sea water that will come in contact with the animals
?. Turn on water
?. Wash hands
?. Dry hands with paper towel
?. Turn off faucet with paper towel
?. Turn on the HEPA filter
?. Wipe surfaces (bench, incubator handles, carboys, etc.) with 70% ethanol before and after working in the room
?. Keep 70% ethanol squirt bottles in a designated space to prevent confusion with ASW squirt bottles
TITLE: Bulk RNA sequencing
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details bulk RNA sequencing from the mouse small intestine.

[STEPS]
SECTION: Procedure
1. Collect the ileum from a PBS-perfused mouse.
SECTION: Procedure
2. Thoroughly flush the ileum sample with cold 1x PBS.
SECTION: Procedure
3. Incubate samples in TRIzol reagent (Thermo Fisher Scientific, Cat #15596026; Waltham, MA) and store at -80 °C until ready for bulk RNA sequencing.

SECTION: Procedure
4. Extract RNA and construct library using Illumina TruSeq chemistry.
SECTION: Procedure
5. Sequence the libraries using an Illumina NovaSeq6000.
SECTION: Procedure
6. Samples are multiplexed in each lane, which yielded targeted number of paired-end, 100bp reads for each sample. RTA (Illumina) for base calling and bcl2fastq2 (version 2.19) are used for converting BCL to fastq format, coupled with adaptor trimming.
SECTION: Procedure
7. Perform a pseudoalignment to a kallisto index created from transcriptomes (GRCm38) using kallisto (0.44.0).
SECTION: Procedure
8. Test differentially expressed genes under various conditions using DESeq2R packages designed to test differential expression between two experimental groups from RNA-seq counts data.
SECTION: Procedure
9. Genes are considered differentially expressed if they had an adjusted p-value <0.05 and a log2fold change below or above 0.5. Normalize the differential expression for each gene.
TITLE: PBMC Thawing Protocol
AUTHORS: Fang Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for thawing peripheral blood mononuclear cells (PBMC). </div><br/><div class = "text-block"><span>For a protocol detailing Culture and Stimulation, please view the following: </span><a href="https://www.protocols.io/private/DEB5A3831911012B2B1FD77561652138" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PBMCs Culture and Stimulation</span></a><span style = ":;">.</span></div><div class = "text-block"><span>For a protocol detailing Cell Staining for Flow Cytometry Assay, please view the following: </span><a href="https://www.protocols.io/private/F887A98B1CA97529FE641462466879C2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cell Staining for Flow Cytometry Assay</span></a><span>. </span></div></div>

[STEPS]
?. Warm Washing buffer and medium to  in a water bath.
37 °C
?. Remove vials from liquid nitrogen and transport them to the lab on dry ice.
?. Thaw frozen vials, only 1 vial at a time, in a  water bath. When cells are nearly completely thawed, carry the vials to the hood and swab them with 70% EtOH.
37 °C
?. Gently remove PBMCs (avoid pipetting up and down, as the cells are very fragile at this stage) and transfer the cells into a 50 mL falcon tube (Fisher scientific #14-432-22) containing .
[warmed washing buffer]
?. Use  to rinse out the cryovial and gently mix the cells by inverting the 50 mL Falcon tube ~5x.
[washing medium]
?. Wash 1: Spin the cells: . Pour off the supernatant.
Centrifuge: 400 34, 8 min
?. Wash 2:Suspend the cell pellet in  (dropped slowly along the side of the tube) and resuspend the cell pellets, add . Spin the cells: .
[prewarmed medium]
[complete medium]
Centrifuge: 400 34, 8 min
?. If cells were thawed in the presence of benzonase, perform an additional wash with culture medium in the absence of benzonase.
?. Count cells and determine viability by Trypan blue staining.