eamag/protocols-with-steps huggingface dataset:
TITLE: Collection of human vagal tissue samples for TEM imaging
AUTHORS: Terry  Powley, Deborah  Jaffey, Anita Gupta, Plamen Mihaylov, John Wo

[DESCRIPTION]
This protocol describes the methods used to generate samples of human vagus tissue suitable for TEM imaging.

[BEFORE_START]
Ensure that all appropriate authorizations and permissions for sampling of human tissue are obtained in advance - see protocol for details.

[STEPS]
SECTION: Sampling
1. Vagus nerve samples were obtained intra-operatively from organ donors. Separate consents for organ donation and research were obtained. Informed consent for study participation was obtained from a parent and/or legal guardian. No organ donors were prisoners. A diagnosis of brain death or circulatory death as appropriate had been previously established by independent medical professionals according to local institutional regulations. All procedures related to tissue procurement and research use for tissues were in compliance with and approved by regulatory oversight review committees at University of Indiana School of Medicine, Indiana Donor Network or the local Organ Procurement Organization, and Purdue University. The vagus nerve biopsies were obtained from a continuous series of qualified organ donors meeting the diagnostic and medical criteria for organ donation and with consent for research participation in place. Donors from whom multivisceral transplants (lungs and stomach) were pursued were excluded from the research studies to enable removal of as complete sets of samples as possible. The research team was not involved with the identification and selection of organ donors.
SECTION: Sampling
2. Age, gender, BMI, race, cause of death, and delay time between death and sample recovery were recorded for all donors/samples. All data was deidentified.
SECTION: Sampling
3. After the completion of transplant organ harvesting, samples of the vagus nerve were collected. Approximately 10–20 mm long segments of the vagus nerve were removed from the following locations:
Cervical – left / anterior
Cervical – right / posterior
High abdominal trunk above hepatic and celiac branches – ventral / anterior
High abdominal trunk above celiac branches – dorsal / posterior
Ventral /anterior gastric branch at the level of the cardia
SECTION: Processing
4. All vagus nerve segments were immediately placed in a fixative solution containing 2% paraformaldehyde and 2.5% glutaraldehyde for 5 days at 4 °C.
  
  
The fixative solution was changed daily and the specimens were kept on an oscillating plate during the immersion fixation process (at 4 °C). The fix solution was always refrigerated and was always less than one week old. 
SECTION: Processing
5. After completion of the fixation process, the tissue specimens were rinsed (3 × 30 min at 4°C) in PBS, transferred to individual shipping vials under PBS, and shipped overnight on ice to the TEM laboratory for further processing and microscopy - see the protocol below:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Table 1: Age distribution of subjects staged for AMD</p>
<p>Table 2: CFI levels in subjects with AMD</p>
<p>Fig. 3: Preincubation of CFI with amyloid B (1-42) reduces CFI bioactivity</p>
<p>Fig. 4: Anti-amyloid B antibody, GSK933776 effectively blocks amyloid B's ability to inhibit CFI bioactivity</p>
<p>Fig. 5:  Measurement of CFI bioactivity</p>
<p>Fig. 6: CFI bioactivity in a cohort of subjects with staged AMD and non-AMD normals</p>
<p>Fig. 8: CFI bioactivity in plasma smaples of Alzheimer's disease</p>

[STEPS]
TITLE: Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach 
AUTHORS: Katie Pollock, Michael Ranes, Ian Collins, Sebastian Guettler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from </span><span style = "font-style:italic;">Escherichia coli </span><span>and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.</span></div></div>

[STEPS]
TITLE: Left Ventricle Contraction Curves
AUTHORS: Giorgio Bozzi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The folder LVOBJEVL.zip contains the LeftVentricle.exe file and the KLVG directory with service files. Unzip the folder.</div><div class = "text-block">LeftVentricle.exe Program</div><div class = "text-block">The LeftVentricle.exe is an example program that requires the use of a graphics tablet for data input. I suggest a Wacom Intuos Pro L or Calcomp Drawing Board III graphics tablet. Some examples are available for those without a graphics tablet.</div><div class = "text-block">The heart, accelerating two masses of blood, must submit to the laws of dynamics. The contraction causes a shortening of the heart, and blood expulsion causes a lowering of the organ by the third principle of dynamics. Since shortening equals lowering, cardiac activity is balanced. Therefore, people do not perceive cardiac contraction despite the intensity of the force developed.</div><div class = "text-block">Left ventricular angiography allows measuring systolic aortic plane lowering, but the projective method alters some values. The most significant approximation concerns the longitudinal axes of the cavity. During diastole, ventricular anatomic axis and angiographic longitudinal axis coincide. No in systole because the cardiac apex remains stationary, whereas the angiographic axis shortens and rises. For this reason, a functional coincidence, we superimpose the end-diastolic and end-systolic longitudinal axes for calculations.</div><div class = "text-block">Read Guidelines </div><div class = "text-block">Finally, click "Calculations" on the strip menu.</div></div>

[STEPS]
?. This Zip LVOBJEVL contains LeftVentricle.exe and a service DIR KLVG.
TITLE: Splenocyte isolation from mice
AUTHORS: sffschn

[DESCRIPTION]
Simple protocol for isolation of spleenocytes from mouse spleen.

[STEPS]
1. Collect spleens in some PBS

2. Place spleen on a 70 µm mesh filter ontop of a 50 mL Falcon 

 

2.1. max. 2 spleens per filter
3. pass through 70 µm mesh filter using a pistol of a 2 mL syringe 
4. wash with FACS buffer and top up to 50 mL 
50 mL 
  
  2 %
  1 mM

5. Spin down for 5 min   
400 x g, 5 min, 4 °C 

 

6. Pour off supernatant 
7. Add 1 mL of RBC lysis buffer per spleen and incubate for 5 min at RT
 1 mL 
5 min 
Room temperature 

8. Add a bit of FACS buffer and pass through new 70 µm mesh filter into a new 50 mL Falcon 
9. Fill up to 50 mL 
50 mL 

10. 400 x g, 5 min, 4 °C 

11. Pour off supernatnat 
12. Resuspend in 5 mL of FACS buffer per spleen 
5 mL 

13. Count cells 
 
  


14. 400 x g, 5 min, 4 °C 

15. Resuspend cells to obtain a concentration of 1 x 10^8 cells/mL 
1x10^8 2147 

16. Continue with   for isolatoin of primary mouse NK cells.
TITLE: Potential determinants of COVID-19 vaccine confidence and receptivity among the primary school’s stakeholders in Bangladesh: A cross-sectional study to assess the effects of education
AUTHORS: dn.roy

[DESCRIPTION]
This method designed to (i) investigate COVID-19 vaccine confidence and receptivity among the primary school’s stakeholders in Bangladesh, (ii) identify the potential determinants of vaccine confidence, and (iii) assess the effects of education on vaccine confidence and receptivity. This cross-sectional study used a bilingual, multi-item, closed-ended, anonymous questionnaire, adopted from the theoretical analysis of relevant literatures on topic. A total of 699 primary school’s stakeholders interviewed purposively via face-to-face approach while 10.3% of them declined to give consent. Binary logistic regressions and Chi-squared tests were employed to rationalize the study outlined objectives. Raw data were inserted into Microsoft Excel version 10 and imported to Statistical Package for the Social Science (SPSS) software. IBM-SPSS version 25 (RRID: SCR_016479)was used for analyzing the data in which p<0.05 was considered significant cut-point value.

[STEPS]
TITLE: Measuring dopamine release in human-derived iPSCs using High-Performance Liquid Chromatography (HPLC) coupled to Electrochemical Detection (ECD)
AUTHORS: Kaitlyn ML Cramb, Stephanie J Cragg, Richard Wade-Martins

[DESCRIPTION]
This protocol allows for the detection of evoked or tonic dopamine release from human iPSC-derived dopamine neurons using High-Performance Liquid Chromatography (HPLC) coupled to electrochemical detection. 

[GUIDELINES]
This assay can be performed on any mature iPSC-derived dopamine neurons (DANs), but success has been particularly found on iPSC-DANs that have been grown in culture for 50-90 days following a modified Krik’s protocol. Optimal density found to be plated at 500 000 / well in a 24 well cell culture dish. Collected samples can be stored long-term at -80oC.

All steps are performed at Room Temperature (RT).

[STEPS]
SECTION: Preparation
1. Add 1 µL of perchloric acid (PCA) to a labelled light-protected 1.5 mL Eppendorf tube for each sample.
SECTION: Preparation
2. Prepare Ringers Buffers for tonic or evoked release (see Materials). Store short-term at 4°C.
SECTION: Dopamine (DA) Release Assay
3. Aspirate media from hiPSC-DANs plated as stated in guidelines. 

SECTION: Dopamine (DA) Release Assay
4. Wash hiPSC-DANs one time with 100 µL phosphate buffered saline (PBS).
SECTION: Dopamine (DA) Release Assay
5. Aspirate PBS from hiPSC-DANs.
SECTION: Dopamine (DA) Release Assay
6. Add 100 µL of Ringer’s Buffer for 5 minutes.
SECTION: Dopamine (DA) Release Assay
7. Collect Ringer’s Buffer in pre-labelled tubes containing PCA.
SECTION: Dopamine (DA) Release Assay
8. Snap freeze on dry ice (optional). 
 

SECTION: HPLC-ECD
9. If snap-frozen and stored at -80oC, thaw samples on ice. 
SECTION: HPLC-ECD
10. Spin samples at 10 000 g for 10 minutes.
SECTION: HPLC-ECD
11. Run samples on HPLC column using a mobile phase running at 1 mL/min on a 4.6 x 150 mm Microsorb C18 reverse-phase column and Decade II ECD with a glassy carbon working electrode (Antec Layden) set at 0.7 V with respect to an Ag/AgCl reference electrode. 
SECTION: HPLC-ECD
12. Calculate concentrate of dopamine released in sample compared to known standards.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This extraction protocol follows the DArT DNA Extraction protocol but contains modifications (including increasing the concentration of PVP to 4 %</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for carrying out RNA Stable Isotope Probing experiments using seawater or vent fluids to examine microbial bicarbonate uptake. It was developed at Axial Seamount to examine autotrophy in deep-sea hydrothermal vent fluids. We have used modified versions in seawater, cold crustal environments, and other hydrothermal vents as well.</p>

[BEFORE_START]
<p>This protocol assumes you have already collected your fluid, eg seawater, vent fluids, etc.</p>

[STEPS]
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TITLE: Single-step purification by heat shock
AUTHORS: Hossein Tarrahimofrad, Amir  Meimandipour, Sareh  Arjmand, Mohammadtaghi  Beigi Nassiri, Ehsan  Jahangirian, Hossein  Tavana, Javad  Zamani, Somayyeh  Rahimnahal, Saeed  Aminzadeh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>After the cultivation period, cells were collected by centrifugation at 3500 × g for 30 min at 4 °C and resuspended in 4 ml lysis buffer (50 mM NaH</span><span style = "vertical-align:sub;">2</span><span>PO</span><span style = "vertical-align:sub;">4</span><span>, 300 mM NaCl, 10 mM Imidazole and 0.05% Tween 20 at a pH of 7). The mixture was sonicated with 4 × 40 sec pulses followed by 20 sec rest between cycles at 4 °C. The crude extracts were then centrifuged at 9000 × g for 30 min at 4 °C, and the resulting supernatant was heat-treated in a hot water bath at 90 °C for 15 min. Insoluble material was separated by centrifugation at 13000 × g for 10 min, and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide. The purified elution fractions were dialysed overnight in a 50 mM potassium-phosphate buffer (pH=7) at 4 °C.</span></div></div>

[STEPS]
?. Cultivation cells  , collected by centrifugation    and resuspended in 4 ml lysis buffer (, ,   and   at )
50 mL
37 °C
Centrifuge: 3500 34, 30 min, 4 10
[NaH2PO4]
[NaCl]
[Imidazole]
[Tween 20]
?. The mixture was sonicated with 4 × pulses   followed by  rest between cycles at .
[Amper 0.7]
4 °C
?. The crude extracts were then centrifuged  and the resulting supernatant was heat-treated in a hot water bath at .
Centrifuge: 9000 34, 4 10
90 °C
?. Insoluble material was separated by centrifugation at , and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide.
Centrifuge: 13000 34, 10 min, 4 10
?. The purified elution fractions were dialysed .
4 °C
TITLE: Acetone extraction of bacteria pellet for HPLC (2022 iGEM)
AUTHORS: Team Fudan iGEM

[DESCRIPTION]
Pre-cold acetone is generally used for protein precipitation. Here, we collect the supernatant for membrane lipid analysis.

[STEPS]
2. Add 4 times the volume -20°C acetone (3 mL in our case) to the extract, and vortex.
3. Incubate sample at -20°C for 1 hour. 60 min 

4. Centrifuge at 13500 rpm for 10 minutes at 4°C. Collect the supernatant without touching the protein pellet.

5. Store the acetone extracted sample at -80°C for long term use, or -20°C for short term use.
1. Grow bacteria in in 150 mL 2xYT media overnight. Harvest by centrifugation at 4000 rpm for 10 minutes. Discard the supernatant.
TITLE: No Title
AUTHORS: 

[STEPS]
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SECTION: Data Analysis
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SECTION: Day Prior to Assay
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SECTION: Materials to Prepare
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span class="cit-title">The following is a modification of a Scherz R., 2001 method for colony embedding. The protocol described here is from:<br /><br />Sarah Piccirillo, <em>et. al. </em><a href="http://www.genetics.org/content/184/3/707" target="_blank">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies</a><em> </em>(2010)<br /></span><em>Genetics</em> <span class="cit-vol">184<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">707</span><span class="cit-sep">-</span><span class="cit-last-page">716</span></span><span class="cit-sep cit-sep-after-article-pages">; </span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.109.113480<br /><br />Please see the <a href="http://www.genetics.org/content/184/3/707" target="_blank">full manuscript</a> for additional details.</span>

[GUIDELINES]
To embed colonies for sectioning, we modified a previously described method (<a href="http://www.genetics.org/content/184/3/707.full#ref-33" target="_blank">Scherz et al. 2001</a>), with a major change being the substitution of Spurr's reagent as embedding medium.<br /><br /><strong>Original method:</strong><br />S<span class="sc">CHERZ</span>, R., V. S<span class="sc">HINDER</span> and D. E<span class="sc">NGELBERG</span>, <span class="cit-pub-date">2001</span> <a href="http://jb.asm.org/content/183/18/5402.abstract?ijkey=f30e1e7779510c765afd0b5a7a58319f2a8c0762&amp;keytype2=tf_ipsecsha" target="_blank">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization</a>. <span class="cit-source">J. Bacteriol.</span> <span class="cit-vol">183</span>: <span class="cit-fpage">5402</span>–5413.

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TITLE: Nuclei Isolation from Human Frozen Liver
AUTHORS: Sanjay Subramanian, Stacey Huppert

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to isolate human liver nuclei from frozen samples in an effort to facilitate human nuclei extraction for use in single nuclear RNA sequencing and single cell ATAC sequencing without the need for fresh samples. This approach enables the ability to acquire signatures from all cell types, even those from fibrotic liver samples. A brief fixation step is included in order to preserve nuclear membrane integrity and prevent nuclear leakage. This protocol has been tested and works in frozen human liver samples weighing approximately 1 gram stored in cryopreservation medium. Single nuclear RNA seqencing was run using the 10x Chromium system.</div><div class = "text-block">This project was supported in part by NIH P30 DK078392 (Gene Analysis Core) of the Digestive Diseases Research Core Center in Cincinnati, R01DK107553, and the Cincinnati Pediatric Cell Atlas Center funded by Cincinnati Children’s Research Foundation to Stacey S. Huppert</div></div>

[STEPS]
?. Thaw small tissue sample from frozen preservation medium at room temperature and wash in 1x PBS
?. Briefly mince tissue into small chunks using a scalpel and/or razor in petri dish on ice
?. Homogenize chunks using Tissue Grinder Dounce with Pestle A in 500μL to 1mL of ice-cold TST buffer
[TST buffer]
1mL Tissue Grinder Dounce comes with two pestles with varying clearance. Pestle "A" is best suited for this protocol
Ensure thorough homogenization, even after solution seems sufficiently aqueous. Quality of fixation depends on achieving full dissociation of cellular membranes
?. Filter suspension through a 70μM cell strainer and bring total volume up to 5mL with ice-cold ST buffer with 0.1% PFA. Ensure solution is mixed well to circulate fix
[ST buffer with 0.1% PFA]
?. Centrifuge nuclei at 500g for 5min at 4 °C
[centrifuge 500g at 4 °C]
?. Remove supernatant and resuspend in 5 mL ST buffer
[ST buffer]
?. Repeat steps 5-6
Final resuspension volume may be greater or less than stated and should be judged accordingly based on pellet size
?. Filter solution through a 40μM cell strainer
?. Analyze nuclei morphology using hemocytometer and Trypan blue
?. Adjust nuclei concentration as needed (700-1200 cells/μL for 10x Chromium system)
1000
TITLE: Изаберите Optimus Gel најбољих за бол у зглобовима по доброј цени!
AUTHORS: Optimus Gel Iskustva

[DESCRIPTION]
Оптимус Гел је нови квалитетан производ који обједињује најновија научна достигнућа која заувек ублажавају и отклањају болове у зглобовима, костима и мишићима! Наручите данас са 50% попуста!

[STEPS]
TITLE: Blunt end ligation
AUTHORS: Keyong Sun

[STEPS]
?. [Prepare DNA Fragment]
Amplify target DNA fragment using high-fidelity DNA polymerase for blunt end fragment
?. [Phosphorylation of Recovered DNA ]
?. [Prepare DNA Fragment]
Purify DNA using gel extraction
?. [Ligation]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is part of the VERVE holiday drive to collect exotic off-the-shelf laboratory recipes. More details <a href="https://www.protocols.io/g/verve-net/news/call-for-recipes" target="_blank">here</a>.<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bypcz36.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/byncz36.jpg" data-ofn="8166277389_dd12010e27_o.jpg" /><br />(Image: <a href="https://www.flickr.com/photos/olfiika/8166277389/in/photolist-drCiL2-iPkjBj-iPgoiK-34mbBq-9822Qm-9sHkbu-sgmPZ-aERRuB-8XLBJL-9dotNr-9hi4fq-8VsxtD-9dUQkw-92Uu8b-4ogndv-mgdyHe-mgdZLa-7f49x1-rQWGpQ-mgdC8k-9c6VpS-fVwqwm-93Fj6t-jx6b6e-54r2yb-qTdHaF-8Z8Mii-dQUZTg-5oQLFq-7sTFJ7-Aq5qA-mgfdTS-qNXZN-9sLYUG-77pXJk-4hNrkp-mgfgzJ-7zWBfb-mgdaxD-mgeUGS-5HmNn7-4BHYoA-8ALftz-vHSQEg-dzS16h-5AJDNS-9wFZKS-6nMY87-8WaiCX-a5USCN" target="_blank">Frosty apple</a>, Olga Filonenko, CC BY-SA)

[GUIDELINES]
<strong>Materials &amp; Buffers:</strong><br />
<ul>
<li>One ice bucket with lid </li>
<li>1 cup dry ice </li>
<li>1 test tube rack</li>
<li>1 apple</li>
</ul>

[STEPS]
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TITLE: Preparing Gene of interest for GateWay cloning (2 step PCR process)
AUTHORS: Johannes Wolfram JWD Debler

[DESCRIPTION]
GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites.
This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS

[STEPS]
SECTION: Primer design
1. There are a few things we need to keep in mind when designing the primers in the wider context of Gateway cloning. The idea is that you create an "entry clone" containing your gene of interest. Ususally INCLUDING the start codon, but EXCLUDING the stop codon. This is because one of the points of Gateway recombination is that you use the same entry clone to shuttle your gene of interest into different destination vectors with different properties (different N- or C- terminal tags). If you included the stop codon you would prevent yourself from being able to attach C-terminal tags. 

Gene specific forward primer (PCR 1): 5'-AA AAA GCA GGC TNN -(15 to 20 bp template specific sequence)-3'

The 'NN' here can be any base. Don't use AA, AG or GA though, as that would introduce an in-frame stop codon! They are inserted to keep the reading frame if a destination vector with an N-terminal tag is used. I use 'CC' in all of my primer design. 


Gene specific reverse primer (PCR 1): 5'-A GAA AGC TGG GTN -(15 to 20 bp template specific sequence)-3'

The 'N' here can be any base, just be careful to not accidentally introduce a stop codon.  I use 'A' in all of my primer design.

attB1 adapter primer (PCR 2): 5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-3'
attB2 adapter primer (PCR 2): 5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3'


This is what we are trying to achieve:

  

  


SECTION: PCR 1 - gene specific PCR
2. 
Use your favourite High Fidelity Polymerase. I have used NEB Q5 and Thermo Fisher SuperFi Mastermix as listed below with great success.

 



 

SECTION: PCR 2 -  GateWay attB1 and attB2 adapter primers
3.  Use PCR 1 product as input for PCR 2.
 
 
 
  
 
 
 
 
 


 
SECTION: Run a gel
4. Run PCR 2 product on a gel to make sure you have a crisp band (and to know if the PCR has worked).
SECTION: Gel extraction
5. Extract the PCR 2 band with your gel extraction kit of choice.
SECTION: Proceed to BP recombination step
6. Gateway BP recombination of attB tailed PCR products into pDONR/zeo