eamag/protocols-with-steps huggingface dataset:
TITLE: The future of feedback: Motivating performance improvement through future-focused feedback --Study 3
AUTHORS: Jackie Gnepp, Joshua Klayman, Ian O. Williamson, Sema Barlas

[STEPS]
?. The procedure is essentially the same as in Study 2, post-only group.  The differences are that (a) there is an additional page of instructions (two versions, for which the results did not differ significantly), which is intended to focus the participants on the goal of promoting improved performance, and (b) the post-feedback questionnaire is shorter.If working with a group, pair members randomly.  Within each dyad, assign roles (Regional Manager or Disctrict Manager) randomly by handing the participant the appropriate envelope of instructions.  In the instructions, the names used for each role were pre-tested to be gender-neutral, so could be used with any participant.  Send each dyad to a private room to complete the procedure, with instructions to open the instructions packet after arriving there.  Each packet includes printed instructions, background information, and the questionnaires.
?.
TITLE: Hepatotoxicity during 6-thioguanine treatment: protocol for a systematic review
AUTHORS: Linea Natalie Toksvang, Rikke Hebo Larsen, Thomas Leth Frandsen, Kjeld Schmiegelow, Cecilie Utke Rank

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Hepatotoxicity was recognised as a complication of 6-thioguanine (6TG) in 1976. Since then, 6-TG associated hepatotoxicity in the form of acute sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), has notably been reported in childhood acute lymphoblastic leukaemia (ALL), whereas chronic nodular regenerative hyperplasia (NRH) has been reported in adults with inflammatory bowel disease (IBD) as well as in childhood ALL. However, SOS and NRH should be considered part of a spectrum of microvascular disorders caused by endothelial injury. Nevertheless, the cellular mechanisms responsible for the sinusoidal damage, being pivotal to their development, remain to be established. 6TG-related SOS and NRH have been hypothesised to be dose-related, since they do not seem to occur with low cumulative doses.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">The recent finding that higher levels of thioguanine nucleotides incorporated into leucocyte DNA (DNA-TGN) correlate to a lower relapse risk, calls for reappraisal of the feasibility of prolonged 6TG treatment for childhood ALL, while avoiding the risk of SOS and NRH.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">Objectives</span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">- Primary objective: To assess the incidence of hepatotoxicity in patients treated with 6TG compared to 6MP or standard care.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">- Secondary objective: To explore if a safe dose of 6TG can be established.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
TITLE: Transformation and Preparation of Chemically Competent Bacillus subtilis cells
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Preparation of Chemically Competent Bacillus subtilis cells]
For 20 ml or 50 ml PARIS-Medium mix:  ABCD1final concentrationStockfor 50 mlfor 20 ml2100 mM potassium phosphate buffer pH 7.0360 mM K2HPO41 M (3.48 g in 20 ml for stock)3 ml1.2 ml440 mM KH2PO40.5 M (1.36 g in 20 ml for stock)4 ml1.6 ml53 mM trisodium citrate0.5 M (0.735 g in 5 ml)300 µl120 µl620 mM Potassium-L-glutamate1 M (4.06 g in 20 ml)1 ml400 µl721 mM MgSO41 M (1.23 g in 5 ml)1050 µl (7x)420 µl (7x)81 % Glucose50 % (10g in 20 ml)1 ml400 µl920 mg/ml L-Tryptophan5 mg/ml (25 mg in 5 ml)200 µl80100.1 % Caseinhydrolysat (DIFCO!)10 % (1 g in 10 ml)500 µl200 µl
ABCD1final concentrationStockfor 50 mlfor 20 ml2100 mM potassium phosphate buffer pH 7.0360 mM K2HPO41 M (3.48 g in 20 ml for stock)3 ml1.2 ml440 mM KH2PO40.5 M (1.36 g in 20 ml for stock)4 ml1.6 ml53 mM trisodium citrate0.5 M (0.735 g in 5 ml)300 µl120 µl620 mM Potassium-L-glutamate1 M (4.06 g in 20 ml)1 ml400 µl721 mM MgSO41 M (1.23 g in 5 ml)1050 µl (7x)420 µl (7x)81 % Glucose50 % (10g in 20 ml)1 ml400 µl920 mg/ml L-Tryptophan5 mg/ml (25 mg in 5 ml)200 µl80100.1 % Caseinhydrolysat (DIFCO!)10 % (1 g in 10 ml)500 µl200 µl
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Inoculation of precultures: Spread B. subtilis from Glycerol stock on LB plate and incubate over night for 37°C or over the weekend at 30°C.
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Resuspend single clones in 5 ml Paris Medium (test tube) on incubator roller or plate washer with 1 ml Paris Medium (saves one day work)
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Inoculation of 2.5 ml Paris Medium (test tube) from pre-culture (wash or liquid-overnight culture) to OD580=0.2
?. [Preparation of Chemically Competent Bacillus subtilis cells]
4h incubation on incubator roller at 37°C
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Centrifuge 1 ml pellet cells in reaction tubes for 1 min at maximum speed. Remove the supernatant.
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Resuspend the pellet in 1 ml Paris medium with 10 % (v/v) glycerol.
?. [Preparation of Chemically Competent Bacillus subtilis cells]
Store 100 µl aliquots at -80 °C
?. [Transformation of Bacillus subtilis ]
Thaw aliquots at 37 °C, add 900 µl Paris medium and 500-1000 ng plasmid DNA (test tube)
?. [Transformation of Bacillus subtilis ]
Incubate 6 h in the incubator roller at 37°C
?. [Transformation of Bacillus subtilis ]
Pellet cells, remove 800 µl, resuspend and plate the rest (for normal transformation on LB+antibiotic)
TITLE: Stereotaxic Surgery for Delivery of Tracers by Iontophoresis
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the delivery of a neuronal tracer using the iontophoretic method. The surgery uses a stereotaxic system to target specific brain coordinates in the mouse.  </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: Consistency in Identity Related Sequential Decisions
AUTHORS: Dikla   Perez, Yael Steinhart, Amir Grinstein, Meike Morren

[DESCRIPTION]
We conducted four lab and online experiments, and a field experiment to test our hypotheses and to rule out an alternative explanation. The design of each of the five experiments consisted of presenting participants with two sequential choices. In our experiments we manipulated the expected visibility of the product involved in the first decision, and evaluating whether the identity type (social or personal) emphasized in the second choice matched the identity type emphasized in the first choice. Emphasizing the same identity in both choices represents consistency in identity-related sequential decisions. Experiments were conducted both in public and in private settings, enabling us to confirm that our results were robust across different levels of decision publicity. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to prepare a 100x BG-11 +N stock.</p>
<p>1x BG-11 media could be created from the stock and supplemental stocks and trace metals could be added afterwards.</p>
<p>This protocol is based on Anne Behle M.Sc. <a href="https://www.protocols.io/view/recipe-for-standard-bg-11-media-gtmbwk6" target="_blank" rel="noopener noreferrer">Reciepe for standard BG-11 media</a> protocol.</p>
<p> </p>
<p>Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as <em>Synechocystis</em> sp. PCC 6803, as described.</p>
<p>Media is usually not suitable for marine cyanobacteria.</p>

[GUIDELINES]
<div>
<p>Always work under sterile conditions when handling sterile media or stocks. Work under the clean bench.</p>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Total DNA extraction from microalgae strain samples using NucleoSpin Plant modified kit (Macherey Nagel)
AUTHORS: Sarah  Romac

[DESCRIPTION]
This DNA extraction protocol allows to get both eukaryotic and prokaryotic DNA from microalgae strains, so the microbiome diversity can be studied in cultures by using this protocol.



[BEFORE_START]
-Clean all the area with DNA away.
-Heat the Incubator to 55°C for lysis.
-Heat buffer PL1 at 50°C for at least 30min before to start the extraction to dissolve SDS precipitant.
-Equilibrate buffer PE to 65°C.
-Proteinase K : add 2500 µl Proteinase K buffer PB to adjust concentration to 20 mg/mL, incubate 1min at RT. Don't vortex! Do aliquots of 250µL. Store at -20°C. Don’t freeze/defreeze aliquots more than 3 times.
-Thaw/prepare Lysozyme (20mg/ml) in TE-Buffer.
-Wash Buffer PW2: add 100ml EtOH 96-100%

[GUIDELINES]
You can perform this protocol in safety conditions on the bench.

[STEPS]
SECTION: Cell Lysis
1. Transfer 2mL from the strain in a sterile 2mL microtube.
Centrifuge 5 min at 5000g to pellet the strain.
Discard the supernatant.
Pellet are ready for extraction or can stored at -20°C until extraction.
SECTION: Cell Lysis
2. Add 400µL PL1 + 25µL proteinase K 20 mg/mL.
Vortex.

If you don't need to extract prokaryotic DNA, skip step 3 et directly go to step 4.
SECTION: Cell Lysis
3. Optionnal : 

If you also want to extract the prokaryotic DNA to study microbiome associated to the eukaryote strain, add 100µl Lysozyme 20mg/mL.
Vortex and incubate for 5 min at room-temperature.
SECTION: Cell Lysis
4. Incubate in the thermomixer 60 min @ 55°C, 900 rpm.
SECTION: Clarification of crude lysate
5. After lysis, short spin the lysate and transfer the lysate on a NucleoSpin Filter column (purple column).
Centrifuge 5min @ 11 000g.
SECTION: Clarification of crude lysate
6. Transfer the eluate on a new sterile 2mL microtube. If a pellet is visible, only transfer the supernatant without touching the pellet.
SECTION: Precipitation/Purification
7. Add 550 µL PC buffer, mix by pipetting up and down at least 5 times.
Transfer all liquid onto a green column (max 650µL).
Centrifuge 1min @ 11 000g. Discard the flow-through.
Repeat this step until all liquid has been loaded on the green column.
SECTION: Precipitation/Purification
8. Add 400µL PW1 to the membrane of the column.
Centrifuge 1min @ 11 000g. Discard the flow-through.
SECTION: Precipitation/Purification
9. Add 700µL PW2 to the membrane of the column.
Centrifuge 1min @ 11 000g. Discard the flow-through.
SECTION: Precipitation/Purification
10. Add 200µL PW2 to the membrane of the column.
Centrifuge 2min @ 11 000g. Discard the flow-through.
SECTION: Precipitation/Purification
11. Put the column on a new sterile 1.5mL microtube correctly labeled (name, date, operator).
SECTION: DNA Elution
12. Add 50µL PE (preheated 65°C) onto the membrane. Incubate 5 min @ 65°C.
Centrifuge 1min @ 11 000g.
SECTION: DNA Elution
13. Add again 50µL PE (preheated 65°C) onto the membrane.
Centrifuge 1min @ 11 000g.
SECTION: DNA Elution
14. After centrifugation, store the DNA at -20°C.
TITLE: Patient PBMC differentiation to macrophages
AUTHORS: Rebecca Wallings

[DESCRIPTION]
Patient PBMC differentiation to macrophages

[STEPS]
1. After cryorecovery, plate PBMCs at a final concentration of 3 x 106/mL in a poly-D-lysine coated, 96-well plate, resulting in 3x105 cells per well. 

Incubate PBMCs  at 37°C for 2-hours to allow adherent cells to adhere. 

Wash cells washed 2 x sterile PBS to wash off non-adherent cells, and incubate in growth media
containing 20ng/mL of human macrophage-colony stimulating factor (hM-CSF) for 5 days, with a full media change on day 3 to replace nutrients.
TITLE: UC Davis - IN-VIVO Glucose-stimulates Insulin Secretion Test
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
An in-vivo glucose-stimulates insulin secretion test is designated to determine alterations in insulin secretion by the pancreas upon a bolus IP Glucose injection.</div></div>

[STEPS]
?. Fast mice for 16 hours by taking away food the day before (3:00pm)
?. The following day, Calibrate the glucose meter according to the manufacturer’s instructions.
?. Deprive mice from water then measure blood glucose level using a glucometer and remove immediately approximately 50 μl of blood from the tail via a tail tip cut and transfer directly onto a sterile 0.5ml microcentrifuge tube containing 2ul Heparin.
?. Centrifuge at 8000rpm for 5min then transfer plasma (supernatant) to a new 0.5ml microcentrifuge tube and freeze at -80°C.
?. Give the mouse an intraperitoneal injection of Glucose (2g/kg) with a 27 G needle
?. Continue to take blood samples from the initial tail cut at 2, 5, 15 and 30 min flowing injection and repeat step3 and 4.
?. NOTE:
At the end of the experiment, wipe tail with 70% alcohol and allow drying. Ensure that blood loss from the tail stopped before placing the animal back to its cage.
TITLE: Diagnostic Restriction Digest
AUTHORS: Brian    Teague

[DESCRIPTION]
In this restriction digest, you'll use an enzyme that cuts DNA to cut your miniprepped plasmid. This can give you some evidence as to whether your plasmid is what you expected or not.

  

You'll also use Benchling to predict the result of your digest -- that way, you can compare your prediction to your actual result.

[STEPS]
SECTION: Perform the diagnostic digest
1. For each miniprep, compute the volume that contains 1 µg of DNA.

SECTION: Perform the diagnostic digest
2. In the PCR tube, mix:
The volume of DNA you computed in step 1, up to a maximum of 5 µL
2 µL of CutSmart enzyme buffer
2 µL of PvuII enzyme
Enough nuclease-free water for a total volume of 20 µL



SECTION: Perform the diagnostic digest
3. Mix by flicking the tube gently, then spin down briefly.
SECTION: Perform the diagnostic digest
4. Incubate your reaction for 60 min at 37 °C

SECTION: Simulate the diagnostic digest on Benchling
9. After your ditest is complete, proceed to analyze your restriction digest using gel electrophoresis. 

 

SECTION: Simulate the diagnostic digest on Benchling
5. While your digest is running, you can use Benchling to simulate your digest. Begin by opening the "Cas9 Plasmid" document (or whichever document your instructor indicates). Then, click the "scissors" icon on the right to open the restriction digest panel.

  

SECTION: Simulate the diagnostic digest on Benchling
6. Pull down the "Enzyme Lists" drop-down and choose "NEB". In the "Find Enzyme" box, type "PvuII" (that's the letters "Pvu" and two upper-case i's). The enzyme list should just show "PvuII" -- click it. 


 
 

SECTION: Simulate the diagnostic digest on Benchling
7. Click the blue RUN DIGEST button at the bottom. The new tab that opens tells you the sizes of the DNA fragments were you to digest this plasmid with those enzyme(s).

  


SECTION: Simulate the diagnostic digest on Benchling
8. There's also a new tab called VIRTUAL DIGEST. Click it. Change Ladder option to "NEB 2-Log", which is the ladder we are using in the lab. Voila, a simulation of the gel you can expect to see when you run the digest for real.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here is a short intro on short read assembly using the SPAdes assembler. </p>
<p> </p>
<p>Some notes on tool installation:</p>
<p>To install SPAdes or other tools into your PATH you may wish to use a package manager called Miniconda. For this go to the website here and download the 64-bit Linux distribution:<br />https://conda.io/miniconda.html<br /># then, once you have located this file on your computer, type <br />bash Miniconda2-latest-Linux-x86_64.sh<br /># and follow the installation instructions. When asked to append the PATH info say "yes".<br /># After this you will need to close your terminal and re-open it before beginning again. Then you should be able to install tools using conda. For the module today we will need to install two tools: the sra-toolkit and an assembler called spades. <br />conda install -c bioconda sra-tools<br />conda install -c bioconda spades</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Sinai SCENT TMC - FFPE Blocking, Sectioning, and TMA Construction
AUTHORS: Sojin Kim

[DESCRIPTION]
FFPE Blocking and Sectioning protocol

[GUIDELINES]
Comply with Universal Precautions when handling all specimens.
Use personal protective equipment according to the institution’s guidelines.


[STEPS]
SECTION: FFPE Blocking and Sectioning
1. Rince the collected tissues in PBS to remove blood. 
SECTION: FFPE Blocking and Sectioning
2. Place tissues in at least 10 volumes of buffered formalin or buffered paraformaldehyde
SECTION: FFPE Blocking and Sectioning
3. Incubate for the necessary fixation time
1. 1-2 mm thick: 2-3 hours RT
2. 5-10 mm thick: 5 hours RT
3. >10 mm thick: 2-3 hours RT with overnight at 4'C
SECTION: FFPE Blocking and Sectioning
4. Rinse tissue twice with PBS, and store at 4'C in 70% EtOH
SECTION: FFPE Blocking and Sectioning
7. Immerse in the 70% EtOH in the storage bucket

SECTION: FFPE Blocking and Sectioning
6. Trim and transfer the tissue in the histology cassettes. Small tissue pieces < 1-2mm should be placed between blue sponges within thecassette.  
SECTION: FFPE Blocking and Sectioning
5. Label sample ID on both the front and side of the histology cassettes
SECTION: FFPE Blocking and Sectioning
8. Drop off the samples at the Biorepository and Pathology CoRE (Mount Sinai)
SECTION: FFPE Blocking and Sectioning
9. Sectioning thickness
- Xenium: 5 um
- Orion: 5 um
SECTION: TMA Construction
10. Donor blocks are selected by the researcher and pathologist based on study criteria.

Exclusive criteria 
1. Artifacts such as folding or tearing 
2. Areas with excessive bleeding 

Inclusive criteria (for Lung tissues)
Alveolar, bronchial, and endothelial, while trying to select the same anatomical regions among the samples. 
SECTION: TMA Construction
11. The region of interests are marked using the NDP.view 2 program and submit the annotated H&E scan to the Biorepository and Pathology CoRE Team at Mount Sinai.
TITLE: Hiseq 2000 Library Construction and Sequencing for RNA Seq
AUTHORS: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Hiseq 2000 Library Construction and Sequencing for RNA Seq</div></div>

[STEPS]
?. Isolate polyA RNA from   of total RNA treated by using .It is best to use up to 50 µg as the use of a lower mass (typically 20 µg) has been insufficient for successful library construction. This can be assessed by running final PCR products on an agarose gel; the library construction is considered to have failed when there was no visible band. It is possible to use less than 20 µg of total RNA when isolation of an important sample yielded low RNA mass but library construction was successful.
20 µg
?. Purified polyA RNA is fragmented in a at for  to 200–300 nt fragment sizes.
70 °C
?. The first cDNA strand is then synthesized with random hexamer primers using the .
?. The second-strand synthesis is performed by incubation with  and  .
?. Short double-stranded cDNA fragments are then purified using one of two methods.
?. Our standard procedure was to use the , whereas for samples with low RNA mass it is better to use .
?. Both methods are then followed by end-repair with Klenow polymerase,  .
?. A single 3′ adenosine (A base) was added to the double-stranded cDNA using and  .
?. The Illumina PE Adapter oligo mix is ligated onto the A base on repaired double-stranded cDNA ends and DNA fragments of a selected size are then gel-purified to make sure the insert size is 200 bp (±10% deviation).
?. Thereafter, libraries were amplified by 15 cycles of PCR with and “indexed” paired-end PCR primers; the prepared libraries were 322 bp long.
?. The amplified libraries were denatured with sodium hydroxide and diluted to  in hybridization buffer for loading into a HiSeq flowcell.
?. Read lengths viewed on the HiSeq platform were predominately 90 bp with a small number of sequences in the 84–87 bp range.
?. Samples are sequenced with paired-end reads, and up to eleven samples can be multiplexed into a single lane of the Illumina Hiseq flow cell. With average run time of three to twelve days depending on read length.
TITLE: Rapid Sequencing gDNA
AUTHORS: Carlos Goller

[DESCRIPTION]
ONT Rapid sequencing kit use in a classroom setting. 

[STEPS]
SECTION: Library Preparation
1. DNA tagmentation

Thaw kit components at Room temperature , spin down briefly using a microfuge and mix by pipetting as indicated below:

Lambda DNA (50 μg/ml): thaw at RT, briefly spin down, mix well by pipetting
Fragmentation Mix (FRA): not frozen, briefly spin down, mix well by pipetting
Rapid Adapter (RAP): not frozen, briefly spin down, mix well by pipetting
Sequencing Buffer (SQB): thaw at RT, briefly spin down, mix well by pipetting*
Loading Beads (LB): thaw at RT, briefly spin down, mix by pipetting or vortexing immediately before use
Sequencing Tether (SQT): thaw at RT, briefly spin down, mix well by pipetting

SECTION: Library Preparation
2. Prepare the DNA in Nuclease-free water
SECTION: Library Preparation
2.1. Transfer ~200 ng  genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube

SECTION: Library Preparation
2.2. Adjust the volume to 3.75 µL  with Nuclease-free water

SECTION: Library Preparation
2.3. Mix by flicking the tube to avoid unwanted shearing
SECTION: Library Preparation
2.4. Spin down briefly in a microfuge
SECTION: Library Preparation
2.5. In a 0.2 ml thin-walled PCR tube, mix the following: 
3.75 µL 200 ng template DNA
1.25 µL  FRA

SECTION: Library Preparation
2.6. Mix gently by flicking the tube, and spin down.
SECTION: Library Preparation
2.7. Incubate the tube at 30 °C  for 1 min and then at80 °C for 1 min . Briefly put the tube on ice to cool it down.