eamag/protocols-with-steps huggingface dataset:
TITLE: Extration of Scapharca broughtonii genomic DNA
AUTHORS: Chang-Ming Bai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to eliminate excessive polysaccharide in </span><span style = "font-style:italic;">Scapharca broughtonii</span><span> tissues during DNA extraction.</span></div></div>

[STEPS]
?. Collect 1 mL haemocyte into a 1.5 mL microcentrifuge tube and centrifuge at 1000 g for 10 min at 4℃, then discard the supermatant and resuspend the pellet with 200 μL PBS.
?. Add 200 μL Buffer AL* and 20μL Rnase A, mix thoroughly by vortexing and put the tube at room temperature for 3-5 min.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Add 20 µl proteinase K*. Mix thoroughly by vortexing, and incubate at 56 ℃ for 15 min. Then add 10% SDS (Sodium dodecyl sulfate) and continue to incubate at 56 ℃ for 15 min.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Add the same volume of colled Chloroform at 4℃ and mix by inverting, then put the tube at room temperature for 10 min.
?. Centrifuge at 12,000 g for 10 min at 4℃ and then transfer the supermatant aqueous phase in a new tube.
?. Add 1/3 volume of absolute ethanol, and then mix by inverting.
?. Pipet 700 µl mixture from step 6 into the DNeasy Mini spin column* placed in a 2 ml collection tube*. Centrifuge at 12,000 g for 30 sec at 4℃. Discard flow-through and collection tube.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Place the DNeasy Mini spin column* in a new 2 ml collection tube*, add 500 µl Buffer AW1*, and centrifuge at 12,000 g for 30 sec at 4℃. Discard flow-through and collection tube.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Place the DNeasy Mini spin column* in a new 2 ml collection tube*, add 500 µl Buffer AW2*, and centrifuge at 12,000 g for 30 sec at 4℃. Discard flow-through and collection tube.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Repeat the step 9.
?. Place the DNeasy Mini spin column* in a new 2 ml collection tube*, centrifuge at 12,000 g for 2 min at 4℃, and then put the spin column at room tempreture for 10 min to dry the DNeasy membrane.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
?. Place the DNeasy Mini spin column* in a clean 1.5 ml microcentrifuge tube and pipet 50 µl Buffer AE* directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 12,000 g to elute.
*: these items were provided by Qiagen DNeasy® Blood & Tissue kit (Catalog no. 69504)
TITLE: Transcardial perfusion of mouse tissues
AUTHORS: Rain Kwan, Courtney Wright, Louise Cottle, Alejandra Rangel, Asheeta Prasad

[DESCRIPTION]
This protocol describes how to perform transcardial perfusion and fixation of mouse brain tissues in preparation for immunohistochemical staining or histology. This process includes lethal overdose of mice with sodium pentobarbitone, occlusion of the descending aorta, followed by transcardial perfusion with phosphate buffered saline to clear blood from the vasculature and paraformaldehyde to fix mouse brain tissues.

[STEPS]
SECTION: Experimental Outline
1. Attach a needle into the peristaltic pump tubing and prime the tubing by filling with Room temperature 1x PBS.
SECTION: Experimental Outline
2. Lethally overdose mice with sodium pentobarbitone (100 mg/kg) via intraperitoneal injection.
 

SECTION: Experimental Outline
3. Working in a fume hood, make a lateral incision just beneath the rib cage, immediately inferior to the xyphoid process.
SECTION: Experimental Outline
4. Cut through the diaphragm to expose the thoracic cavity.
SECTION: Experimental Outline
5. Cut through the rib cage on the lateral edge, and then reflect the sternum above the head and hold in place with a pair of forceps.
SECTION: Experimental Outline
6. Complete occlude the descending aorta using a clamp lacking teeth.
 

SECTION: Experimental Outline
7. Insert the gavage needle attached to the pump tubing into the apex of the heart, advancing  -  into the tissue.

SECTION: Experimental Outline
8. Perfuse the tissues for 5 min with Room temperature 1x PBS.

SECTION: Experimental Outline
9. Crimp the tubing to prevent air bubbles entering the line and then switch the source solution for the perfusion pump from Room temperature1x PBS to ice cold 4% PFA in 1x PBS.
SECTION: Experimental Outline
10. Re-start the perfusion pump at 5mL/minute and perfuse the tissues for a further 8 min.
 

SECTION: Experimental Outline
12. Excise the brain and place immediately in a 50 mL sample container filled with ice-cold 4% PFA in 1x PBS. Place sample container on vertical rocker at 4 °C for up to 1440 min.
SECTION: Experimental Outline
13. Discard PFA solution into appropriate waste disposal stream.
SECTION: Experimental Outline
7.1. Clamp the heart and enveloped needle with forceps.
 
 
SECTION: Experimental Outline
7.2. Cut the right atrium and turn on the perfusion pump at a rate of approximately 5mL/minute.
SECTION: Experimental Outline
11. Collect and store PFA run-off for disposal.
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Recovering skeletons
?. 
SECTION: Rinsing
?. 
SECTION: Cleaning
?. 
SECTION: Cleaning
?. 
SECTION: Diluting
?. 
SECTION: Rinsing
?. 
SECTION: Preparing for imaging
?. 
SECTION: Drying
?. 
SECTION: Imaging
?.
TITLE: PROTAC diastereomer Design（negative control）
AUTHORS: boc.protac  

[DESCRIPTION]
Diastereomers are stereoisomers in which molecules have two or more chiral centers and the relationship between molecules is not mirrored. In PROTAC drug development, in order to evaluate the pharmacokinetics of a single diastereomer or mixture of diastereomers, manufacturers should quantitatively analyze samples from the early stages of drug development for a single diastereomer in vivo. This will allow the assessment of the possibility of mutual transformation as well as the distribution of absorption, distribution, biotransformation and excretion (ADBE) of individual isomers. When the drug is a raceme and the pharmacokinetic characteristics of the isomers are different, the manufacturer should monitor the diastereomer separately to determine properties such as dose linearity and the effects of metabolic or excretory changes and drug-drug interactions.

As a leading service provider in drug discovery and development, BOC Sciences is fully qualified and committed to providing one-stop PROTAC®development services, which has become a promising strategy in the field of small molecular drug discovery. With a comprehensive and advanced platform, we design PROTAC diastereomer to customers around the world to meet new drug discovery goals.

[STEPS]
TITLE: Acid Wash Protocol (Alegado Lab)
AUTHORS: Ashleyolguin

[DESCRIPTION]
This protocol guides lab members through the acid wash process 

[STEPS]
SECTION: Pre-requisites (BEFORE proceeding to acid washing)
1. Check acid solution levels

 --> Note: If it's only 1/3 full, make new solution, you will use almost all of the current stock.  

SECTION: Pre-requisites (BEFORE proceeding to acid washing)
3. Gather bottles to be washed and take over to acid washing lab 
SECTION: Pre-requisites (BEFORE proceeding to acid washing)
2. Check to see if Alegado Acid Wash Bin is free or if it is being used

  

SECTION: Acid Washing
4. Fill bin with 10% Hydrochloric acid solution about ½ to 2/3 full and submerge bottles in bin.
SECTION: Acid Washing
5. Fill bottles with acid solution, cap and place in bin.
SECTION: Acid Washing
6. Cover bin with lid. 

--> Note: insure cover is secured.
SECTION: Acid Washing
7. Let sit for minimal of 2 hours. 

--> Note: place bin on Acid wash rack. 
SECTION: Acid Washing
8. Rinse with miliQ water
  

SECTION: Acid Washing
9. Dry on designated drying surface
  

SECTION: Acid Washing
10. Empty bin and rinse with miliQ water.
SECTION: Acid Washing
11. Dry 
SECTION: Neutralization of Acid
12. Plug the drain and test the seal using water 
SECTION: Neutralization of Acid
13. Use acid rinse until the sink fills 3/4 maximum
SECTION: Neutralization of Acid
14. Add sodium bicarbonate
SECTION: Neutralization of Acid
15. Test the pH using a test strip

If the measured pH is between 5.5 and 8.5, unplug the drain and discharge. Repeat the procedure for any remaining acid rinsing.
If the pH is below 5.5, add more sodium bicarbonate
If the pH is above 8.5, add more acid. 

Always test the pH before discharging !
TITLE: Fasting Review
AUTHORS: Ana Priscila Eleodoro Rosa, João Paulo Souza, Vick Nogueira Pileggi, Fernando Bellissimo-Rodrigues, Leonardo Moscovici

[DESCRIPTION]
A structured, overarching, systematic approach to the body of evidence on the
effects of fasting in human health is lacking. This systematic overview of
literature aims to map the existing body of evidence, initiate a living
repository of evidence, and guide the development of a suite of specific
systematic reviews on the effects of fasting on human health. This protocol
describes the methods that will be used to conduct a systematic overview of the
literature on the effects of fasting on human health. The 2020 PRISMA Statement
provides reporting standards for the overview. We expect to develop a
high-level, narrative summary of the body of evidence related to fasting and
its effects on human health. The main limitations of the evidence included in
the overview will be discussed. Limitations of the review processes used in
this overview will also be addressed. High-level implications for practice,
policy, and future research will be described.

[STEPS]
TITLE: URMC TriState SenNet TMC Mouse Sacrifice and Organ Harvest
AUTHORS: Gagandeep Kaur, Irfan Rahman

[DESCRIPTION]
This protocol outlines the method used to sacrifice and harvest the mouse lung tissue to perform downstream analyses for the TriState SenNet TMC Biospecimen Core at the University of Rochester, as part of the Cellular Senescence Network Program (SenNet). 
Care was taken to reduce the pain and distress caused to the mice by performing all the procedures after anesthetising the mice.

[STEPS]
1. Sodium Pentobarbitol ( 60-90 mg/kg of body weight) was administered intraperitoneally to anaesthetize the mice prior to organ harvest.
2. After 5 min of drug administration, the animal response was assessed by pinching the mouse's foot with forceps and observing the withdrawal response/pedal reflex.
3. Upon successful anesthetization, the animals abdomen and chest was cut open and mice was exsanguinated by cardiac puncture.
4. Thereafter, the thoracic cavity was cut open to harvest the mouse lung. 
5. The harvested organ was stored in PBS and kept on ice to perform future assays/ assessments.
TITLE: BioRad Trans-Blot Turbo: fast set-up with own materials
AUTHORS: Karla LH Feijs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is our labs' fast protocol for BioRad Trans-Blot Turbo with our own materials. This works well for many applications, however, for high molecular weight proteins wet transfer conditions remain superior.</div></div>

[STEPS]
?. Prepare all materials, such as transfer buffer, nitrocellulose membrane and WYPEALL cut to the size of the gel. Run a standard SDS-PAGE.
?. Soak the gel in transfer buffer for 10 minutes. In meantime, prepare a stack of 6 WYPEALL papers on the bottom cassette and add the nitrocellulose membrane on top.
?. Place the gel on top of the membrane and add another 6 WYPEALL papers. In general: the lower percentage gel, the better the transfer will be.
?. Use the provided roller to remove any air bubbles with a gentle roll, while applying equal pressure.
?. Close the cassette and place it in the machine. Select the BioRad standard "High MW" protocol, which runs at 1.3A for 10 minutes.
?. Open the cassettes and place the stacks of WYPEALL in a container with fresh transfer buffer. These stacks can be re-used when kept in a sealed container at 4ºC.Discard the gel, or to analyse transfer efficiency place in coomassie staining, and continue with your standard western blotting protocol.Rinse out the bottom cassette with ddH2O, wipe off the bottom cassette with a wet cloth.
TITLE: Measurement of Left Ventricular Performance in Langendorff Perfused Mouse Hearts
AUTHORS: E. Dale Abel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">This protocol describes the procedure used by the DiaComp for measuring left ventricular performance in isolated retrogradely perfused mouse hearts.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bihw4kcn.jpg" /></div></div>

[STEPS]
?. [Langendorff Heart Perfusion Protocol]
Hearts are isolated and the aorta is cannulated using a 20g steel cannula. Hearts are perfused at a constant pressure of 60 mmHg by an aortic cannula delivering warm (37°C) Krebs buffer containing (in mM) 118 NaCl, 4.7 KCl, 25 NaHCO³, 1.2 MgSO4,  1.2 KH2PO4, 2 CaCl² gassed with 95% O², 5% CO². Hearts are perfused with glucose 11mM as sole substrate or in combination with 1 or 1.2 mM palmitate. The pulmonary artery is transected to facilitate coronary venous drainage. A left ventricular polyethylene apical drain is inserted through a left atrial incision to allow thebesian venous drainage. Left ventricular pressure is monitored from a water-filled balloon placed through the left atrial appendage and connected to a Millar transducer. The volume of the balloon is adjusted to obtain a left ventricular diastolic pressure of 7 mmHg. Heart rates are adjusted to 360 beats/min by pacing at 6 Hz at the level of the atria.
?. [Inotropic stress protocol]
After 30 min stabilization, data are acquired under baseline conditions (buffer calcium concentration =2 mM). The hearts are then switched to a second buffer containing 4 mM CaCl². Contractile parameters are again measured after 20 min of stabilization. The langendorff protocols yield the following parameters:(1)  Left ventricle systolic pressure (LVSP): Units mmHg.
(2)  Left ventricle developed pressure (LVDP or LVDevP), which is LVSP – LV
       Diastolic pressure: Units mmHg.
(3)  Heart Rate (HR): Units beats per minute
(4)  Rate Pressure Product (RPP), which is LVDP x HR. Units mmHg/sec
(5)  dP/dtmin and dP/dtmax, which are the maximal rates of LV pressure decay and LV,        pressure development respectively. Units mmHg/msec.(6) Coronary Flow (ml/min). This is determined by measuring the coronary effluent from the perfused heart.
?. [Myocardial oxygen consumption (MVO2).]
Coronary effluent is sampled from the pulmonary artery using a capillary tube. Oxygen content was measured using an Oxygen foxy probe (OceanOptics) and calculated using the formula:Where O² solubility and O² density are 23.9 μl/ml and 0.03933 μmol/μl respectively in a solution at 37°C respectively.Cardiac efficiency is calculated as the ratio of RPP/MVO² expressed as a percentage.
TITLE: Propidium Iodide Cell Cycle Staining Protocol
AUTHORS: Sam Li

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

[STEPS]
?. Harvest cells in the appropriate manner and wash in PBS.
?. Fix in cold 70% ethanol (do not make this with PBS as it can cause protein precipitation during fixation). Add dropwise to the cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping.
?. Fix for at least 30 minutes at 4°C. Specimens can be left at this stage for several weeks at -20°C.
?. Spin at 2000 rpm and be careful to avoid cell loss when discarding the supernatant, especially after spinning out the ethanol. Resuspend in 1xPBS, spin at 2000 rpm for two rounds of washes.
?. To ensure that only DNA is stained, treat cells with Ribonuclease. Add 50µl of 100µg/ml RNase.
?. Add 425µl of Cell Staining Buffer (Cat# 420201) and 25 µl of Propidium Iodide Solution (Cat# 421301).Tip: Do not wash off PI after staining,
TITLE: Growth Curve Stress Test (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for 
 


[STEPS]
SECTION: Prepare 2x synthetic media
1. Put 200 mLof deionized water in a 250 ml bottle. Then add:
0.855 g  
0.335 g   
2.5 g   
5 mL 100X uracil solution (2 mg/mL  )
5 mL 100X leucine solution (12 mg/mL   )

SECTION: Prepare 2x synthetic media
2. Autoclave 121 °C for 30 min 

SECTION: Prepare the yeast cultures
4. The afternoon or evening before lab, prepare a test tube with 5 ml 2xSDC media for each yeast strain you will be testing. 
SECTION: Prepare 2x synthetic media
3. Cool, then add 50 mL 40 Mass / % volume gluco  using aseptic technique.

SECTION: Prepare the yeast cultures
5. Pick a colony from each strain's plate and resuspend it in the media.
SECTION: Prepare the yeast cultures
6. Grow   at 30 °C, either on a roller drum (preferred) or on an orbital shaker at  .

SECTION: Prepare the yeast cultures
7. The next morning, several hours before class, measure the OD600 of each culture.
SECTION: Prepare the yeast cultures
8. Prepare another test tube with 5 ml of 2xSDC media for each yeast strain. Subculture the overnights to an OD600 of 0.1. Place the tubes back in the incubator or shaker.
SECTION: Prepare the yeast cultures
9. Optimally, grow the cultures for at least 3 hours before lab.

 

SECTION: Instructor Tips &amp; Common Student Errors
10. Instructor Tips
This assumes that, once you or your students have identified yeast colonies with successful knockouts, you have struck them out on plates
Log-phase growth is the most reproducible growth mode for yeast. This is why the experiment should start in log phase and why doubling time should be computed in log phase.
Growing the cells in double-strength media means that they can be diluted 1:1 and still be in "complete" media for the stress experiment.
Yeast cells are heavy and settle rapidly toward the bottom of a test-tube. Make sure you vortex them before measuring OD600; vortex again before subculturing; and vortex again before putting them in an assay plate.
Computing doubling time is not optional! Because we're looking at an exponential phenomenon, a small difference in starting concentration is rapidly amplified and can "look" on a graph like a strain is growing slower.
I thought everyone graduated highschool with a basic ability to use a spreadsheet? I am often surprised by the number of students that have trouble with "make a graph of this data."
SECTION: Instructor Tips &amp; Common Student Errors
11. Common student errors
Not computing doubling time to compare growth, but just drawing conclusions from eyeballing a graph.
Not vortexing the yeast cells before putting them in the assay plate.
General data manipulation and interpretation difficulty
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. Here, we describe a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second “template-switch” primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.</p>

[STEPS]
TITLE: Very low-density lipoprotein receptor (VLDLR)-C-tag purification from HEK293E cells
AUTHORS: Andreas Bracher, Patricia Yuste-Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to purify recombinant Very low-density lipoprotein receptor (VLDLR)-C-tag protein from HEK293E cells.

[STEPS]
SECTION: VLDLR-C-tag expression
1. Express VLDLR-C-tag in HEK293E cells cultured in FreeStyle 293 Expression Medium for 5760 min

SECTION: VLDLR-C-tag expression
2. Centrifuge culture and keep conditioned medium.
SECTION: VLDLR-C-tag expression
3. Dialyze 300 mL conditioned medium   against 10 L Binding buffer.

SECTION: CaptureSelect C-tag affinity chromatography
4. Load dialyzed conditioned medium onto a CaptureSelect C-tag affinity column previously equilibrated with binding buffer (column volume, CV: 4 mL slurry for 300 mL dialyzed media) by gravity flow at 4 °C.

SECTION: CaptureSelect C-tag affinity chromatography
5. Wash the column with 5 CV of Binding buffer.
SECTION: CaptureSelect C-tag affinity chromatography
6. Elute VLDLR-C-tag protein with 6x 1 mL of Elution buffer. Collect fractions of 1 mL.

SECTION: CaptureSelect C-tag affinity chromatography
7. Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
 

SECTION: CaptureSelect C-tag affinity chromatography
8. Pool fractions containing VLDLR-C-tag protein.
SECTION: CaptureSelect C-tag affinity chromatography
9. Exchange protein buffer to Binding buffer with a NAP-25 desalting column previously equilibrated with Binding buffer.
SECTION: CaptureSelect C-tag affinity chromatography
10. Collect the fractions containing protein, concentrate by ultrafiltration to >1 mg mL-1, aliquot and flash-freeze purified VLDLR-C-tag in liquid nitrogen for storage at -70 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is short tutorial on one way to calculate dN/dS ratios between pairs of protein-coding nucleic acid sequences using codeml in the PAML package.</p>
<p> </p>
<p>Code is intended for use on an Ubuntu 16.04 LTS OS, but it may work on other Unix or Unix-like systems.</p>
<p> </p>
<p>The programs used in this tutorial are:</p>
<p><strong>codeml in the PAML package</strong>. On a Ubuntu 16.04 LTS system it should be able to install this tool with "sudo apt install paml". http://abacus.gene.ucl.ac.uk/software/paml.html</p>
<p><strong>PAL2NAL.</strong> This is essentially a PERL script that you will want to have handy, either by putting it in the folder that you are working in or by putting it somewhere that is in your PATH. http://www.bork.embl.de/pal2nal/</p>
<p><strong>clustal-omaga</strong>. You should be able to install this with "sudo apt install clustalo" This is a nice amino acid and nucleic acid alignement program. For purposes here your choice of aligner is not critical, so if you prefer MAFFT or Muscle or something else you can continue using those. http://www.clustal.org/omega/ </p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?.