eamag/protocols-with-steps huggingface dataset:
TITLE: Measurement of heat production (thermogenesis) in cells using ERthermAC dye_microplate method
AUTHORS: Chih-Hao Wang, Yu-Hua Tseng, Yu-Hua Tseng

[STEPS]
?. Wash differentiated adipocytes with PBS twice
?. Add 100 μL of staining solution per well of a 96-well plate
?. Incubate 37oC for 30~45 min
?. Wash with PBS twice
?. Add 90 ul of DMEM-H serum-free and phenol red-free medium each well
?. Equilibrate plate at 25 oC for 15 min
?. Measure 2-3 points of basal red fluorescence (ex/em, 543/590) in a microplate reader
?. Add 10 uL of medium or 10 mM CL-316,243 (CL)/forskolin (Fsk) each well (10x dilution, final 1 μM)
?. Measure the red fluorescence (ex/em, 543/590) every 5 min up to 1.5 ~ 2 hr
?. Calculate the decrease of fluorescence over time after drug treatment
TITLE: Cloning individual AAV capsid variants
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 the procedure to clone individual AAV capsid variants for single variant characterization.

[STEPS]
1. Digest   with   and   



2. Design 100 bp primers for the desired plasmid variant that cover the insertion region with ~20 bp overlap of the backbone
3. To synthesize the double-stranded DNA fragment, anneal the primers by PCR with 20 cycles of 98 °C for10 s, 60 °C for 30 s and 72 °C for 10 s using   

4. Assemble the variant-specific fragment into the digested backbone by Gibson assembly
5. Transform the assembled plasmid into   

6. Select colonies on carbenicillin/ampicillin-LB-agar plates.
TITLE: Non-Volant Mammal Measurements - ISL Peru
AUTHORS: Cristian Tirapelle, Gideon Erkenswick, Mrinalini Watsa, Pamela Sánchez-Vendizú

[DESCRIPTION]
This protocol describes the external morphological measurements taken on mammals prior to release and is actively used by Field Projects International at the Estación Biológico Los Amigos, Madre de Dios, Peru.


 


[GUIDELINES]
This protocol accompanies the "Handling and Sampling Mammal - ISL Peru" protocols.

[STEPS]
SECTION: Lenghts
1. Head length: Distance from the tip of the nose until the occipital bone. 

 


SECTION: Lenghts
2. Head width: it is measured on the widest part of the cranium. As a reference, we measure the distance in between the base of the pinnae. 
 

SECTION: Lenghts
3. Body length: is intended as the length of the truck of the body. It is measured from the shoulder blades to of terminal tail vertebra.
SECTION: Lenghts
4. Total length: Distance from the tip of nose to of terminal tail vertebra. 

 



SECTION: Lenghts
5. Tail length: it is measured from dorsal flexure at base of the tail to tip of last vertebra. 
SECTION: Lenghts
12. Testis and Vulva lengths: The greatest distance along the longitudinal axis.
SECTION: Lenghts
13. Testis width The greatest distance along the transversal axis.
SECTION: Circumferences
14. All the circumferences are measured only in medium to large mammals and with measuring tape
SECTION: Circumferences
15. Waist circumference: The smallest circumference of the abdomen taken close to the pelvis.
SECTION: Circumferences
16. Chest circumference: The widest part of the ribcage.
SECTION: Circumferences
17. Neck circumference: It is measured at the mid-neck level.
SECTION: Circumferences
18. Arm circumference: It is measured at the mid part of the arm
SECTION: Circumferences
19. Forearm circumference:  It is measured at the widest part of the forearm
SECTION: Circumferences
20. Thigh circumference: It is measured at the mid part of the thigh.
SECTION: Circumferences
21. Calf circumference: It is measured at the widest part of the calf.
SECTION: Lenghts
6. Femur length : it refers to the length of the thigh, it is measured using the greater trochanter and the bent knee (included) as the reference points, and by connecting the two with measuring tape. 
SECTION: Lenghts
7. Tibia length:  it refers to the length of the calf, it is measured using the bent knee joint and the bent tarsal joint (included) as the reference points, and by connecting this the two with measuring tape.
SECTION: Lenghts
8. Humerus length:  Distance from the shoulder joint till the bent elbow joint included.
 

SECTION: Lenghts
9. Forearm length: Distance from the elbow joint to the carpal joint included, using a measuring tape.
 

SECTION: Lenghts
10. Hindfoot length: Distance from proximal margin of calcaneus to tip of the longest claw.
SECTION: Lenghts
11. Forefoot length: Distance from proximal margin of the wrist to tip of the longest claw.
TITLE: Test protocol II
AUTHORS: Abby   Moore

[DESCRIPTION]
This is a test protocol

Here's an protocol reference:


 


Here's a citation:


 


[BEFORE_START]
This is what you should know before you start


 


[GUIDELINES]
Responsibilities....

[STEPS]
1. Use 80:20 MeOH:H2O for this step. This is not easy to access by machine.
2. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

3. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

4. Use this piece of equipment:

 


5.
TITLE: Growth curve analysis
AUTHORS: Sebastiaan Kuiper

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>To observe the potential of defense mechanisms of either native or synthetic systems in </span><span style = "font-style:italic;">Escherichia coli  </span><span>(and more) when incubated with a bacteriophage stock. </span></div></div>

[STEPS]
?. [Preparations]
Media and bacteriophage stock solutions :1L Luria-Bertani (LB) media (with antibiotics)Desired Bacteriophage stock solution in LB media (with known Plaque Forming Units (PFU) ml-1)
?. [Preparations]
Fill in plate reader protocol as follows:Set temperature: 37°C    preheat before moving to next stepStart kinetics:         Runtime 15:00:00 (HH:MM:SS), Interval 0:04:00Shake:         medium, 0:30 (MM:SS)Read:         Absorbance Endpoint, Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msecEnd kinetics    preheat before moving to next stepStart kinetics:         Runtime 15:00:00 (HH:MM:SS), Interval 0:04:00Shake:         medium, 0:30 (MM:SS)Read:         Absorbance Endpoint, Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msecEnd kinetics        Runtime 15:00:00 (HH:MM:SS), Interval 0:04:00Shake:         medium, 0:30 (MM:SS)Read:         Absorbance Endpoint, Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msecEnd kinetics        medium, 0:30 (MM:SS)Read:         Absorbance Endpoint, Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msecEnd kinetics        Absorbance Endpoint, Full Plate        Wavelengths: 600        Read Speed: Normal, Delay: 100 msec
?. [Preparations]
Prepare overnight cultures of desired samples (with associated antibiotics).
?. [Plate reader]
Measure OD600 of overnight cultures and dilute cultures to an OD600 of 0.02
?. [Plate reader]
Load  into a            Include a serie of LB (without bacteria) as a control and as zero point for the OD600 measurements!
[of diluted overnight culture]
?. [Plate reader]
Start plate reader protocol  and let the bacteria grow to an OD600 of 0.11.
?. [Plate reader]
Prepare Bacteriophage PFU dilutions (with associated antibiotics) for;     MOI 101 :  4.0 x 1010 PFU ml-1    MOI 100 :  4.0 x 109   PFU ml-1    MOI 10-1 :  8.0 x 108  PFU ml-1    MOI 10-2 :  8.0 x 107  PFU ml-1    MOI 10-3 :  8.0 x 106  PFU ml-1An OD600 of 0.10 correlates to 8.0 x 108 cells per ml.The above concentrations are required when 20 μl of bacteriophage dilution is added into 180 μl of cell culture with an OD600 of 0.11 (1:10 dilution).
?. [Plate reader]
At the moment an OD600 of 0.11 is reached, the plate reader must be stopped and  must be added to a final volume of  to both the samples and the LB controls.     * include as a control, a serie without bacteriophages and only LB media (with antibiotics)
[of bacteriophage dilution*]
200 µl
?. [Plate reader]
Restart the plate reader protocol and measure over 15 hours the growth of the samples.
TITLE: Organization for Lab Data using Bash
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Organization is important in the IPA and MSBIL</div></div>

[STEPS]
?. [Introduction]
Organization is crucial in Bash and in our lab. Bash operates through directories, therefore we must be precise when storing raw data, intermediate steps, and finished images. *NOTE: Never process data in the raw data directory. Copy the raw data to a new directory in case issues occur. Always keep the original data!
?. [HPC]
Log onto the HPC web interface using the https://dx.doi.org/10.17504/protocols.io.7ezhjf6 protocol.If you are using the linux box, then follow the https://dx.doi.org/10.17504/protocols.io.62whgfe protocol.
?. [Steps]
For our lab all of our data is to be precisely organized with in  the data folder. Here, things will further be broken down based upon project, development, ect...it is important that you know where the data set you are working on is to be stored. If you are unsure, ask someone to avoid later confusion.This protocol will follow an example project "TEST" so that it is easy to see how to organize your files.
?. [Steps]
Go to the lab's research group if you are not already currently there:cd /rsgrps/hutchinsone
?. [Steps]
Check your working directory:pwd
?. [Steps]
Going into the Data filecd Data/
?. [Steps]
If you need to know the files under your current directory, type:lsThis should list all the contents in your current location. We will go into projects for this example.cd Projects/
?. [Steps]
If the data you are working with is for a new project or does not associate with any other pre-existing data, you may want to create a new directory.mkdir EXAMPLE
?. [Steps]
Go into your new directory and add these new directories.cd EXAMPLE/mkdir TESTmkdir Raw
?. [Steps]
Copy your raw data from the hutchinsone/Data/Raw directory to your new Raw file within your project.cd rsgrps/hutchinsone/Data/Rawcp make_believe_data.tif /rsgrps/hutchinsone/Projects/EXAMPLE/Raw
?. [Steps]
From here you may want to think about how to further organize your data.  For example:Sample nameEx. VivoIn. VivoControlInjury typeContrast agents/dyesImage processing stepsFinal Images
?. [Closing Remarks]
As stated before organization of your data is important. Especially with our type of data where it is easy to mix up files, and it is hard to figure out the sorting later. Any information you can include about your data in titles or the in folder names should be done to avoid later confusion.If you are confused about the Bash commands used in this protocol or want to know more, please refer to this cheat sheet https://github.com/LeCoupa/awesome-cheatsheets/blob/master/languages/bash.sh
TITLE: Phylogenetic tree and ancestral sequence reconstruction
AUTHORS: Matthew Kellom

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the alignment, tree building, and ancestral sequence reconstruction of metagenomic protein coding sequences.</div></div>

[STEPS]
?. Obtain protein-coding metagenome FASTA files.
?. Obtain a set of amino acid sequences that are known/trusted target homologs (Uniprot works well for this).
?. Use DIAMOND (blastx can also be used but will be slower for large datasets) to search for target hits from within the metagenome sequences. Recommend an e-value cutoff of at least 1e-20.
?. Translate protein-coding target hits to amino acid sequences with TranslatorX.
?. Use DIAMOND (or BLAST) to annotate hits against a local copy of the NCBI nr database.
?. Choose taxa of interest, and select sequences of appropriate length for the protein from the metagenome hits.
?. Align the collected sequences and closely related complete outgroup sequence(s) with MAFFT. Recommend settings of --maxiterate 1000 --localpair
?. Make tree with RAxML. Recommend settings -f a -k -m PROTGAMMAAUTOInspect the tree to see if it makes sense. If a node looks out of place, consult the alignment and figure out why and if you want to keep it in the dataset.
?. Check RAxML amino acid substition model and branch support with IQTree. Recommend settings -m TEST -nt AUTO -alrt 10000
?. Branch support with MR Bayes. Will need to create a nexus and mbatch file. Recommend mbatch settings:lset nst=6 rates invgamma;prset aamodelpr=fixed(lg); (The model choice here should match the model that RAxML and IQTree select.mcmcp ngen=1000000 nruns=2 nchains=4 samplefreq=100 printfreq=10000 relburnin=yes burninfrac=0.25;mcmc;sumt;sump;
?. Submit the MAFFT alignment and RAxML best_tree to the FastML web portal (http://fastml.tau.ac.il/). You can use the local version of this but I found it easier to just use the web portal. Be sure to select Amino Acids and the correct model from the tree building.  Use Gamma Distribution and Maximum Liklihood. Deselect Optimize Branch Lengths and joint reconstruction.
?. Obtain the 25 most probable ancestral sequences at the nodes of interest from the FastML results page and visualize the tree results in FigTree.
TITLE: Murashige and Skoog (MS) agar
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Murashige and Skoog medium</span><span> (or </span><span style = "font-weight:bold;font-style:italic;">MSO</span><span> or </span><span style = "font-weight:bold;font-style:italic;">MS0</span><span style = "font-weight:bold;font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">(MS-zero)</span><span>) is a </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">plant</span></a><span style = ":;:;"> </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">growth medium</span></a><span> used in the laboratories for cultivation of plant </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">cell culture</span></a><span>. MSO was invented by plant scientists </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Toshio Murashige</span></a><span> and </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Folke K. Skoog</span></a><span> in 1962 during Murashige's search for a new </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">plant growth regulator</span></a><span>. A number behind the letters MS is used to indicate the sucrose concentration of the medium. For example, MS0 contains no sucrose and MS20 contains 20 g/l sucrose. Along with its modifications, it is the most commonly used medium in </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">plant tissue culture</span></a><span> experiments in laboratorium.</span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;vertical-align:super;">[1]</span></a></div><div class = "text-block">Source:</div><div class = "text-block">http://cshprotocols.cshlp.org/content/2010/2/pdb.rec12142.full?text_only=true</div></div>

[STEPS]
?. Add Murashige and Skoog Basal Salt medium to a 1L flask
4 g
?. Add dH2O
800 ml
?. Add  of Bacto Agar to flask and autoclave
7 g
?. Add dH2O up to 1L
?. Adjust the pH to 5.7 using 2 N Potassium hydroxide KOH
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background: </strong>Selective reporting distorts the aggregate body of scientific evidence, wastes resources and can harm patients’ health and the credibility of science. Selective reporting may result from a focus on preferred findings by researchers and others stakeholders. However, it is unclear if some persons or environments are at greater risk of selective reporting than others. Our review assesses what is known and what is postulated about determinants of selective reporting in the scientific domain.</p>
<p><strong>Methods/design:</strong> Using search terms for bias and selection combined with terms for reporting and publication, we systematically search the PubMed, Embase, PsycINFO and Web of Science databases. A 25 percent random selection of records is reviewed for inclusion by at least two reviewers based on title and abstract if available and, if needed, the full text. Inter-rater agreement is calculated. Examining the content of the entire article, including, for example the discussion section, we extract phrases mentioning determinants. From this, we compile a structured list of possible determinants. The results are also categorized by type of source (empirical result or view, study design, and academic discipline if variability suffices). We follow the PRISMA-P and PRISMA reporting guidelines for systematic reviews where applicable.</p>
<p><strong>Discussion:</strong> We use principles of the systematic review along with principles of qualitative content analysis to focus on the nature of possible determinants of selective reporting. Our review includes both empirical findings and theoretical considerations. It will identify priority questions for further research on mechanisms of selective reporting. Furthermore, it will help in assessing risk of selective reporting. Such “risk profiling” may inform effective policy development on responsible conduct of research. The approach of combining quantitative and qualitative assessment techniques is expected to optimally inform well-targeted intervention studies and inspire policy development.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
This protocol comes from a group of other protocols. This protocol is (4) of (4): <br />1. <a href="https://www.protocols.io/view/Large-Volume-Marine-Cyanophage-Phage-Protocols-c3iykd" target="_blank">'Large Volume Marine Cyanophage Phage Protocols'</a><br />2. <a href="https://www.protocols.io/view/DNA-Extraction-Protocol-c32yqd" target="_blank">'DNA Extraction Protocol'</a><br />3. <a href="https://www.protocols.io/view/DNA-Precipitation-Protocol-c34yqv" target="_blank">'DNA Precipitation Protocol'</a><br />4. <a href="https://www.protocols.io/view/Checking-DNA-Concentration-with-Agarose-Gel-c35yq5" target="_blank">'Checking DNA Concentration with Agarose Gel'<br /><br /></a>Needed:<br />
<ul>
<li>0.7% Agarose</li>
<li>(Agarose)</li>
<li>(TAE Buffer)</li>
<li>(HINDIII (lambda) DNA)</li>
<li>Nanodrop</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Strep pull down assay
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
Strep pull down assay for FIP200(NTD)-TSF WT or mutants were co-transfected with GST-ATG13(363-517) and/or ULK1(MIT)-MBP 




[STEPS]
SECTION: Expression
1. Transfect 10 ml HEK GNTi cells at concentration of 2 × 10^6 cells/ml
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Expression
3. In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
4. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C

SECTION: Expression
5. Add mixture to cells. Let cells grow for 2880 min

SECTION: Expression
6. Harvest Cells 500 rpm, 4 °C, 10 min

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at-80 °C until purification. 

SECTION: Pull down
8. Homogenize the pellets in 0.5 ml of lysis buffer/protease inhibitors/1% TritonX-100, and clarified after 40000 x g , 15 min.  

SECTION: Pull down
10. Wash the beads four times, and eluted in 50 μl lysis buffer/4 mM desthiobiotin. 







SECTION: Pull down
11. Mix 18 μl eluent with lithium dodecylsulfate (LDS)/BME buffer, heated at 60 °C for5 min and subjected to SDS/PAGE gel. The gel was then stained with Coomassie brilliant blue G250. 




SECTION: Pull down
9. Incubate the lysate with 30 μl Strep-Tactin Sepharose resin (IBA-Lifesciences) at 4 °C for 180 min
TITLE: T Cell Activation with anti-CD3 Antibodies Protocol - Mouse
AUTHORS: Sam Li

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

[STEPS]
?. Prepare a 5µg/ml solution of anti-CD3ε (clone 145-2C11) in sterile PBS.
?. Dispense 50µl of the antibody solution to each microwell of the 96-well assay plate. For the unstimulated control wells, add 50µl of sterile PBS.
?. Seal plate. Incubate at 37°C for 2 hours or 4°C overnight.
?. Aseptically decant antibody solution from microwell plate.
?. Wash plate microwells 3 times with sterile PBS. Discard liquid.
?. Prepare single cell suspension of cells of interest.
?. Resuspend cells in supplemented cell culture medium to 1-2 x 106/ml.
?. Aliquot 200µl cell suspension into plate microwells. Cover with lid. Incubate at 37°C in 5% CO2 and 100% humidity for 3 days.
TITLE: RNase One Ribonuclease digestion
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Modified from: http://www.promega.com/paguide/chap6.htm#RNaseAmount</div><div class = "text-block">This protocol was used to remove RNA from DNA preparations.</div><div class = "text-block">The RNases commonly used in an RPA are RNase I , RNase T1 or a combination of RNase T1 and RNase A. RNase I cleaves after all four ribonucleotides, RNase T1 cleaves after G residues and RNase A cleaves after A and U residues. For most efficient cleavage of the single-stranded regions immediately adjacent to the double-stranded RNA, we recommend RNase I (RNase ONE™ Ribonuclease, Cat.# M4261). </div><div class = "text-block">Use 1–10u RNase ONE™ Ribonuclease per 10μg of total RNA or 0.1–1u of RNase ONE™ Ribonuclease per 1μg of poly(A)+ RNA.</div></div>

[STEPS]
?. Incubate 5–10μg of total NA 85°C for 5 minutes to denature the NAs.
[total NA]
85 °C
?. Add 300μl of RNase digestion buffer and the appropriate amount of RNase ONE™ Ribonuclease (1-10u per 10μg of total RNA). Incubate the samples for 30–60 minutes at 20–37°C.
[RNase digestion buffer]
20 °C
?. Stop the reaction as follows: Add 10μl of 20% w/v SDS and 2.5μl of 20mg/ml proteinase K. Incubate for 15 minutes at 37°C.
[20% w/v SDS]
37 °C
[20mg/ml proteinase K]
?. Add 825μl of ice-cold 100% ethanol, and chill at –20°C for 30 minutes, then centrifuge at maximum speed in a microcentrifuge for 15 minutes at 4°C to pellet the RNA.
[ice-cold 100% ethanol]
-20 °C
4 °C
?. Resuspend the pellet.
?. Carefully remove the supernatant. Wash the pellet using 300μl of 70% ethanol and dry.
[70% ethanol]
?. Extract once with phenol:chloroform:isoamyl alcohol, and remove the aqueous phase to a clean microcentrifuge tube containing 1μl of 10mg/ml carrier tRNA .
[10mg/ml carrier tRNA]
A yeast tRNA is an effective coprecipitant to aid in recovery of small amounts of nucleic acids.