eamag/protocols-with-steps huggingface dataset:
TITLE: A protocol for tissue clearing and three-dimensional imaging of human sigmoid mucosal biopsies 
AUTHORS: Pu-Qing Yuan, tao li, Yvette Taché

[DESCRIPTION]
This protocol was developed for tissue clearing and 3D imaging of human sigmoid mucosal biopsies by adapting and modifying the original CLARITY tissue clearing technique/immunofluorescence protocols that we established for 3D imaging and phenotyping of human colonic enteric nervous system. By using this new protocol, a piece of entire human sigmoid mucosal biopsy specimen with the volume about 6x3x1-2 mm (length x wide x thickness) can be completely cleared within 3-4 days. The 3D images and videos generated from cleared biopsy samples showed clear spatial views of nerve innervation, distributions of mast cells and enteric glial cells, and configurations of mast cells with nerve fibers in the sigmoid colonic mucosa.

[STEPS]
1.
TITLE:  Isolation of bacterial DNA with Gentra Puregene kit (modified protocol)
AUTHORS: o.pogoutse, Megan Frederickson

[DESCRIPTION]
The Qiagen protocol for purification of genomic DNA from gram-positive bacterial cultures using Yeast/Bact. Kit ( Gentra Puregene Handbook - QIAGEN) was modified to aid in the isolation of bacterial DNA from heterogeneous, low volume, low OD  cultures

[STEPS]
SECTION: Cell lysis
1. transfer culture to 1.5ml microfuge tube
SECTION: Cell lysis
2. centrifuge for 10min at 12000rpm to pellet cells
SECTION: Cell lysis
3. decant or aspirate off supernatant
SECTION: Cell lysis
4. add 1 ml sterile PBS to pellet and vortex briefly to resuspend cells
SECTION: Cell lysis
8. resuspend pellet in 50ul PBS buffer
SECTION: Cell lysis
9.  add 2ul of MetaPolyzyme solution (5mg/ml) (Sigma#MAC4L)
SECTION: Cell lysis
10. incubate overnight at 35C
SECTION: Cell lysis
11. add 310ul lysis solution (Qiagen #158113) to each tube. Pipette up and down gently to
 resuspend cells

SECTION: RNase treatment
13. add 1.2ul of 10mg/ml RNase A per tube
14. invert tube gently to mix
15.  incubate at 37ºC for 30-60 minutes  
SECTION: Protein precipitation
16. cool sample to room temperature
SECTION: Protein precipitation
17. add 105ul of protein precipitation solution (Qiagen #158123) 
SECTION: Protein precipitation
18.  invert tube gently several times to mix
SECTION: Protein precipitation
19. place on ice for 30 minutes
SECTION: Protein precipitation
20. centrifuge at 12000rpm for 3 minutes.
(The precipitated protein should form a tight pellet).
SECTION: Protein precipitation
21. transfer supernatant to a fresh sterile tube (by pipette)  
SECTION: DNA precipitation
22.  add 1-2ul of glycogen (20mg/ml) (Thermofisher #R0561) to  the supernatant and mix it by pipetting up and down
SECTION: DNA precipitation
23. add 400ul of 100% isopropanol
SECTION: DNA precipitation
24.  invert tube gently 50 times to mix 
(optionally: incubate at RT for 30 min to improve DNA yield)
SECTION: DNA precipitation
25. pellet at 13000rpm for 3 minutes
SECTION: DNA precipitation
26. carefully decant off isopropanol (the pellet might be loose)
SECTION: DNA precipitation
27.  add 500ul of 70% ethanol (cold), and invert tube a few times to wash the DNA pellet
SECTION: DNA precipitation
28. centrifuge at 13000rpm for 3 minutes
SECTION: DNA precipitation
29. pour off ethanol carefully – pellet may be loose
SECTION: DNA precipitation
30.  air dry pellet for 15-30 minutes  
SECTION: Cell lysis
12. heat samples to 80ºC for 5-10 minutes to complete the lysis
**stop step: lysed cells are stable at room temperature 

SECTION: DNA precipitation
31. rehydrate DNA in ~35-50ul of 10mM Tris buffer (pH7.5-8.0)
 (adjust for large or smaller pellet)
SECTION: Cell lysis
5. centrifuge for 10 min at 12000rpm
SECTION: Cell lysis
6. decant of supernatant
SECTION: Cell lysis
7. repeat  steps 4-6  one more time
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Components:<br /></strong><br />10 mM K-MES, pH 6.2, 100 mM KCl, 45 mM MnCl<sub>2</sub><sup>.</sup>4H<sub>2</sub>O, 10 mM CaCl<sub>2</sub><sup>.</sup>2H<sub>2</sub>O, 3 mM HaCoCl<sub>3</sub>. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: HMA determination
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol is used to determine the yield of HMA producted by E. coli by using HPLC. 

[BEFORE_START]
Wash the sample bottle with ddH2O for 3 times and  absolute ethanol for 1 time by using ultrasonic cleaner.
Prepare 8 mM KH2PO4 (pH 2.4) and filter it before use.

[STEPS]
SECTION: Activation of the cells
1. Inoculate 5 ml of LB medium with 1% volume of E. coli culture and culture at 37℃ for 12h.  

 

SECTION: Induction of protein expression
2. Prepare ZYM-5052 medium (5ml) by mix ingredients below: (for detailed recipe   

 

SECTION: Induction of protein expression
3. Inoculate 5 ml of ZYM-5052 medium with 1% volume of E. coli LB culture and culture at 37℃ for 12h.  

SECTION: Fermentation
4. Prepare M9 medium (10ml) by mix ingredients below:

 

SECTION: Fermentation
5. Centrifuge 6OD bacteria in 1.5 ml eppendorf (EP) tube at 4,200 x rpm for 10 minutes at room temperature.
SECTION: Fermentation
6. Decant or aspirate and discard the culture media.
SECTION: Fermentation
7. Use 200μl  M9 medium to spirate and dis completely resuspend cell pellet and culture at 37℃ for 12h.  

SECTION: Sample preparation
8. Centrifuge at 15,000 x g  for 10 minute at room temperature.
SECTION: Sample preparation
9. Take 100 µl of supernatant into another EP tube and mix it with 900μl ddH2O.

SECTION: Sample preparation
10. Filter the sample with 0.22μm membrane filters and inject the sample into a clean sample bottle.
SECTION: HPLC (high performance liquid chromatography)
11. HMA was measured an HPLC (Agilent 1260) equipped with a UV detector and a Welch Xtimate C8 column (4.6 × 250 mm, 3.5 µm). The samples were analyzed under the following gradient of eluent A (8 mM KH2PO4, pH 2.4) and eluent B (100% acetonitril): 5 min 10% B, 20 min linear gradient to 30% B, 3 min linear gradient to 50% B, 5 min 50% B, 2 min linear gradient to 10% B, 5 min 10% B. HMA was detected at 215 nm.
TITLE: Gibson Assembly Cloning 
AUTHORS: Claire Y Chiang, Suzanne R Pfeffer

[DESCRIPTION]
Gibson assembly requires a vector backbone and one or more inserts that have been PCR amplified. The inserts should have 15-20 base pairs of overlap at ligation sites, so primers used to amplify the inserts should contain a tail overhang with this homology. The vector backbone should be linear.

A Gibson reaction uses a master mix that can be homemade or commercially purchased. Add the appropriate amount of master mix, then your vector and insert at 2-3 fold molar excess for a 20 µl reaction. Exact ratios will need to be optimized based on the size of your vector and inserts and how many inserts you have. Incubate this reaction at 50°C for 60 minutes. Then transform this reaction into DH5 alpha (or competent cell of choice) and plate on LB agar with appropriate antibiotic.







[STEPS]
SECTION: Prepare Gibson master mix
1. Make 5 ml of 5x reaction buffer
SECTION: Prepare Gibson master mix
2. Prepare master mix

SECTION: Prepare Gibson master mix
1.1. 25% PEG-8000		        1.25 g
500 mM Tris-HCl pH 7.5	2.5 ml
50 mM MgCl2		        0.25 ml of 1M
50 mM DTT		                0.25 ml of 1M
1 mM each dNTPs	                0.05 ml each of 100mM
5 mM NAD		                0.5 ml of 50 mM
SECTION: Prepare Gibson master mix
3. Use master mix as 1.5x. Snapfreeze in 15 µL aliquots and store at -80 °C.

SECTION: Prepare Gibson master mix
2.1. Master Mix
320 µl 	5X Reaction Buffer (above)
0.64 µl 	10U/µl T5 exonuclease
20 µl	2U/µl Phusion
160 µl	40U/µl Taq Ligase
700 µl	Water
SECTION: Gibson assembly
4. Prepare linearized vector backbone by restriction enzyme digestion or PCR amplification.
SECTION: Gibson assembly
5. Prepare inserts by PCR amplification. Ensure that each insert has 15-20 base pairs of overlap with other inserts/vector at ligation sites, added by primer design.
SECTION: Gibson assembly
6. Take a 15 ul aliquot of 1.5x Gibson master mix on ice. Add 50-100 ng of vector with 2-3 fold molar excess of PCR inserts, and sterile water if necessary, to make total reaction volume up to 20 µL.

SECTION: Gibson assembly
7. Mix well and let incubate in PCR machine for 60 min at 50 °C

SECTION: Plasmid preparation
8. After incubation is complete, transform into competent cells (DH5α) and plate onto LB agar with appropriate antibiotic. Let this grow at 37 °C 60 min.

SECTION: Plasmid preparation
9. Pick a single colony to grow in 5 ml LB culture at 37 °C   and purify plasmid to check sequencing.
TITLE: TS Spurrs - cell pellet (TM - 013, sections 5.3 &amp; 6.6)
AUTHORS: sandra.crameri

[DESCRIPTION]
This method is used for conventional processing of cell pellets to Spurrs resin.

[GUIDELINES]
All time are minimum times, it is acceptable to go over time specified for any given step. Good place steps to leave overnight or at 70% ethanol and 50/50 Spurrs/Ethanol mix.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:
SECTION: CONVENTIONAL
2. 2.5 % volume   for at least 40 min 

SECTION: CONVENTIONAL
3. Wash 0.1 Molarity (M) Sorenson's Phosphate Buffer pH 07.2 (300mosmol/kg) for 15 min 

SECTION: CONVENTIONAL
4. 1 % volume   in buffer for 60 min 

SECTION: CONVENTIONAL
5. 70 % volume   for at least 20 min 

SECTION: CONVENTIONAL
6. 95 % volume   for 20 min 

SECTION: CONVENTIONAL
7. 100 % volume   for 20 min 

SECTION: CONVENTIONAL
8. 100 % volume   dried molecular sieved for 20 min 

SECTION: CONVENTIONAL
9. 50 % volume   50 % volume   for 30 min 


SECTION: CONVENTIONAL
10. 100 % volume   30 min 

SECTION: CONVENTIONAL
11. 100 % volume   60 min 

SECTION: CONVENTIONAL
12. Oven polymerise overnight at 65 °C
TITLE: Working Alone in the Lab--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Working alone in the laboratory should be minimized; however, CHEM 584 students can work alone in the laboratory if they adhere to the following Standard Operating Procedure (SOP).</div><div class = "text-block">Also, the doors to the laboratory must remain closed during times when the class is not in a scheduled session.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<p>This protocol is from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<p> </p>
<p>Please see the manuscript for the full method details.</p>
</div>
</div>

[BEFORE_START]
<p>You'll need: </p>
<p> </p>
<p><strong>RIPA buffer: </strong></p>
<ul>
<li>50mM Tris-HCl (pH 8)</li>
<li>150mM NaCl</li>
<li>1% NP-40</li>
<li>0.5% sodium deoxycholate </li>
<li>0.1%SDS</li>
</ul>
<p> </p>
<p><strong>5 ml 0.32 M sucrose buffer: </strong></p>
<ul>
<li>320 mM Sucrose</li>
<li>1. 5 mM MgCl<sub>2</sub></li>
<li>1 mM EGTA</li>
<li>10 mM HEPES (pH 7.5)</li>
</ul>
<p> </p>

[GUIDELINES]
<p>Primary antibody dilution used: Guinea pig anti-m/ratFwe2 (GP67), 1:500; Rabbit anti-SVP38, 1:1000 (Sigma); Rabbit anti-GM130, 1:5000 (Abcam); Mouse anti-Tubulin, 1: 10000 (Sigma); Mouse anti-α-Actin, 1:10000 (Sigma). Secondary HRP-conjugated antibodies were diluted in 1:5000 (Jackson ImmunoResearch).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for cell adhesion assays using 293T cells or N-cadherin-deficient 293NC cells (Yamagata et al., 2018).  HEK293T(ATCC CRL-3216) cells are easily transfectable with plasmids.  The 293NC cells lack N-cadherin (Cadherin-2), but maintain the property of its parental 293T cells.  Thus, this 293T variant is useful to test cell adhesion (Goodman et al., 2016; Yamagata et al., 2018).   Here, I describe a protocol and some technical tips.  </p>
<p>  </p>

[BEFORE_START]
<p>293NC cells are available upon request to Joshua Sanes (sanesj@mcb.harvard.edu).  </p>
<p> </p>
<p>Center for Brain Science,</p>
<p>Department of Molecular and Cellular Biology,</p>
<p>Harvard University</p>
<p>52 Oxford Street, Cambridge, MA 02138 USA</p>

[GUIDELINES]
<p>Technical inquries related to this protocol should be directed to Masahito Yamagata, PhD</p>
<p>(e-mail: yamagatm@mcb.harvard.edu or yamagatm2@gmail.com). </p>
<p> </p>
<p>Center for Brain Science,</p>
<p>Department of Molecular and Cellular Biology,</p>
<p>Harvard University</p>
<p>52 Oxford Street, Cambridge, MA 02138 USA</p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Abstract</strong></p>
<p><strong>Background</strong></p>
<p>This paper describes the association between workplace violence and job satisfaction among physicians and nurses in Macau. It further examines the risk factors for intrinsic and extrinsic job satisfaction among these professionals.</p>
<p><strong>Methods</strong></p>
<p>Convenience sampling was sourced from six health centers supervised by the Macau Health Bureau, namely, the Coloane Health Center, Coloane Shek Pai Wan Temporary Health Center, Taipa Elderly Macau University of Science and Technology Hospital, Macau Sino-Portuguese Nurses Association, Macau Association of Medical Volunteers and the Macau Surgical Association. <strong>Design</strong>: This study uses a cross-sectional self-administrative survey. <strong>Instruments:</strong> The study used case studies research instruments for workplace violence in the health sector by country (from the ILO, ICN, WHO, PSI) and the Minnesota Satisfaction Questionnaire and Perceived Stress Scale. The data collection period spanned from August to December, 2014. <strong>Data analysis</strong>: Multiple logistic regression examines levels of intrinsic and extrinsic satisfaction in physicians and nurses and significant correlates affecting their job satisfaction.</p>
<p><strong>Results</strong></p>
<p>A total of 720 (14.9% physicians) participants were recruited. Worry about WPV, on-call duty and shift work, experience of bullying and verbal abuse and employment sector emerged as significant correlates affecting the intrinsic and extrinsic job satisfaction of physicians and nurses. Working on the frontline, being aged 30 to 39, coming from an ethnic minority, and perceived stress were significant correlates affecting nurses’ job satisfaction.</p>
<p><strong>Conclusions</strong></p>
<p>WPV remains a significant concern in healthcare settings in Macau. Healthcare professionals should deploy protective strategies to combat the negative psychological effects of WPV. Stakeholders should legally enforce a zero-tolerance policy towards WPV within healthcare workplaces. The perpetrators of WPV should be told that violence will be deemed a violation of human rights and not tolerated, not least on account of how WPV is detrimental to healthcare professionals’ mental wellbeing, risking irreversible physical and psychological harm for its victims.</p>
<p><strong>Keywords: </strong>Workplace Violence, Physicians, Nurses, Job satisfaction, Occupational health, Macau</p>

[STEPS]
TITLE: Chimeric Protein-AG and Protein-LAG sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA is used to study the interaction between protein-AG (SpAG) and protein-LAG (PLAG) with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA is used to study the interaction between protein-AG (SpAG) and protein-LAG (PLAG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled PLAG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.28.
TITLE: PrediKt Study Protocol
AUTHORS: Calvin Wang

[DESCRIPTION]
We strive to build a predictive model to estimate risk of postoperative knee pain in patients undergoing total knee replacement.

[STEPS]
SECTION: PrediKt Study Protocol
1. Select patients from Zuckerberg San Francisco General Hospital and Trauma Center, specifically looking for patients who underwent total knee arthroplasty. Use inclusion and exclusion criteria to select the sample size of patients in our retrospective cohort.
SECTION: PrediKt Study Protocol
2. Extract the Pua and Sanchez-Santos predictive models from their respective papers by analyzing the results section and odds ratio coefficients. Using each model, fit it against the retrospective patient data as directly as possible, making note of any model variables that cannot be directly mapped to our data.
SECTION: PrediKt Study Protocol
3. Analyze the degree to which the Pua and Sanchez-Santos models accurately predicts our data using C-indices and R2-indices.
SECTION: PrediKt Study Protocol
4. Create an aggregated list of patient demographic and preoperative variables that have been identified as post-TKA PPSP. Analyze our retrospective patient data to determine which of these variables can be accounted for or approximated by the available data.
SECTION: PrediKt Study Protocol
5. Perform a univariable and multivariable logistic regression and determine significant predictors of PPSP. If appropriate, create a nomogram prediction model for PPSP using our regression data, and analyze the accuracy of our model using key measurements (e.g., AUC, Brier score, ROC, sensitivity, specificity).
SECTION: PrediKt Study Protocol
6. Enhance our prediction model using supervised ML techniques (e.g., decision tree, K-nearest neighbor, random forest, support vector machine) and determine the diagnostic accuracy for each of these model optimizations.
SECTION: PrediKt Study Protocol
7. Repeat step 6 with unsupervised ML techniques.
TITLE: P2 20/05
AUTHORS: Maria  Gul

[DESCRIPTION]
qa

[STEPS]
1. test 

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Nam libero tempore, cum soluta nobis est eligendi optio cumque nihil impedit quo minus id quod maxime placeat facere possimus, omnis voluptas assumenda est, omnis dolor repellendus. Morbi imperdiet, mauris ac auctor dictum, nisl ligula egestas nulla, et sollicitudin sem purus in lacus. Nam quis nulla. Nunc auctor. Fusce wisi. Aliquam erat volutpat. Curabitur ligula sapien, pulvinar a vestibulum quis, facilisis vel sapien. Cras pede libero, dapibus nec, pretium sit amet, tempor quis. Nullam dapibus fermentum ipsum.
2. test 2 

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Nam libero tempore, cum soluta nobis est eligendi optio cumque nihil impedit quo minus id quod maxime placeat facere possimus, omnis voluptas assumenda est, omnis dolor repellendus. Morbi imperdiet, mauris ac auctor dictum, nisl ligula egestas nulla, et sollicitudin sem purus in lacus. Nam quis nulla. Nunc auctor. Fusce wisi. Aliquam erat volutpat. Curabitur ligula sapien, pulvinar a vestibulum quis, facilisis vel sapien. Cras pede libero, dapibus nec, pretium sit amet, tempor quis. Nullam dapibus fermentum ipsum.
TITLE: Association between the overall inflammatory potential of diet during pregnancy and the child’s subsequent risk of type 1 diabetes
AUTHORS: Rohina Noorzae, anne.ahrendt.bjerregaard, tih, sfo

[DESCRIPTION]
A growing body of evidence suggests that diet is a modifiable determinant of low-grade systemic inflammation, as expressed by inflammatory markers. Processed meats, dairy products, and refined carbohydrates have been linked to higher levels of pro-inflammatory markers(1,2). Contrastingly, certain types of fruits, vegetables, and whole grains have been associated with lower levels of inflammation and inflammatory diseases (2–4).
Type 1 diabetes, an autoimmune disease, is due to both genetic and environmental contributions, resulting in an immune-mediated destruction of insulin-producing β-cells and leading to lifelong insulin treatment (5). The incidence of type 1 diabetes is highest in countries following a Western lifestyle (6), and the increasing rate has been faster than can be accounted for by genetic drift, therefore pointing to the importance of environmental factors (6). As an autoimmune response against the insulin-producing beta cells is a central feature of type 1 diabetes (2,7,8), external factors that may influence the immune system and inflammatory responses constitute potentially interesting candidate exposures for novel research. Since the immune system establishes and develops in early life and to a certain degree antenatally (7), there is good reason to explore the role of diet consumed during pregnancy with a special focus on dietary components with inflammation- and immuno-regulatory properties. It is possible that such components may alter the immune cell reactivity and inflammatory state of the mother during pregnancy and subsequently in the child (9,10).
Several validated indexes have been developed to quantify and reflect the overall inflammatory potential of diet by correlating the frequency of consumption of food groups with the concentration of inflammatory markers in the blood (11,12). Such inflammatory diet scores have been associated with an increased risk of cardiovascular disease (13), colorectal cancer (14), and prostate cancer (15).
Our study will address this question: Is there an association between inflammatory dietary patterns during pregnancy and type 1 diabetes risk in the offspring? The hypothesis of this study is that consuming a pro-inflammatory diet during pregnancy increases offspring risk of developing type 1 diabetes. This will be studied in a prospective cohort design in the Danish National Birth Cohort (DNBC), where we have developed an inflammatory diet index for pregnancy diet using the methodology previously described by Tabung et al. to rank the women with respect to the overall inflammatory potential of the diet they had reported to consume during pregnancy (16).

[STEPS]