eamag/protocols-with-steps huggingface dataset:
TITLE: Agarose Gel DNA Extraction (QIAquick)
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extract DNA from agarose gel using Qiagen® QIAquick® Gel Extraction Kit. The kit can be used for centrifuge processing or vacuum processing. The steps given here are only for centrifuge processing.</div><div class = "text-block">Link to the original product website is provided in the Exernal Link field. PDF file of full protocol from manufacturer is attached (available online). I do not claim any credit for the development of the kit or protocol. Please refer to the original protocol provided by Qiagen for any clarifications.</div></div>

[STEPS]
?. [Excise Gel]
Label and weigh 1 uncolored 1.5 mL centrifuge tube for each gel band to be exracted.
?. [Excise Gel]
Using a clean razor blade, excise DNA band from gel and place in corresponding 1.5 mL centrifuge tube. Try to cut the gel as close to the DNA band as possible
It is critical to avoid exposure of skin or eyes to UV irradiation. When using UV light source for fluorescence ensure that all skin is covered. Wear a full-face mask made of a UV blocking material such as polycarbonate. Be aware of others around you to avoid accidentally exposing them to UV irradiation.
It may be best to use a dulled razor blade when cutting to avoid damage to transilluminator surface.
?. [Excise Gel]
Weigh the 1.5 mL tube with the DNA gel slice, and determine weight of gel.
Subtract the tube weight found in step 1 to detemine gel weight.
?. [Excise Gel]
Add 3 volumes Buffer QG to 1 volume gel. For >2% agarose gels, add 6 volumes Buffer QG instead of 3.
gel ~
?. [Dissolve Gel]
Incubate at  for  or until gel has completely dissolved.
50 °C
?. [Dissolve Gel]
Vortex every 2-3 min to help dissolve gel.
?. [Dissolve Gel]
After gel slice has dissolved, check the color.If color is orange or violet: add ,  , and mix.
10 µl
[sodium acetate]
?. [Dissolve Gel]
Add 1 volume isopropanol to the tube and vortex mix.
?. [Bind and Spin DNA]
Place a QIAquick spin column in a provided 2 mL collection tube.
?. [Bind and Spin DNA]
Apply the sample to the QIAquick column and centrifuge for  .
If volume exceeds   , add and centrifuge only    first, then add and centrifuge the remaining volume.
?. [Bind and Spin DNA]
Discard flow-through and place the QIAquick column back into the same tube.
?. [Bind and Spin DNA]
Add  Buffer QG to the QIAquick column and centrifuge for  . Discard flow-through and place back into the same tube.
500 µl
?. [Wash DNA]
Add  Buffer PE to QIAquick column and centrifuge for  . Discard flow-through and place column back into the same tube.
750 µl
?. [Elute DNA]
Place QIAquick column into a clean 1.5 mL centrifuge tube.
?. [Elute DNA]
Add  Buffer EB or Nuclease Free Water to the center of the QIAquick membrane.
50 µl
For increased DNA concentration add   instead.
?. [Elute DNA]
Centrifuge the column for  .
?. [Elute DNA]
Let stand for  to increase DNA yield.
?. [Elute DNA]
Discard the QIAquick column, and store DNA in microcentrifuge tube or aliquot to smaller volume.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Teeth were first disinfected with 75% ethanol and then washed with phosphate-buffered saline. Briefly, PDLSCs were separated from periodontal ligament in the middle one-third of the root. Subsequently, MSCs were digested in a solution of 3 mg/mL collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) and 4 mg/mL dispase (Roche Diagnostics Corp., Indianapolis, IN, USA) for 1 h at 37°C. Single-cell suspensions were obtained by cell passage through a 70-μm strainer (Falcon, BD Labware, Franklin Lakes, NJ, USA). MSCs were grown in a humidified, 5% CO<sub>2 </sub>incubator at 37°C in DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 2 mmol/L glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). The culture medium was changed every 3 days. MSCs at passages 3-5 were used in subsequent experiments. Human embryonic kidney 293T cells were maintained in complete DMEM with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. </p>

[STEPS]
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TITLE: Western blotting for Rubicon and Pacer expression
AUTHORS: Dan Tudorica

[DESCRIPTION]
Traditional.

[STEPS]
1. Wash adherent cells to be analyzed twice with PBS, then scrape into microcentrifuge tubes. Pellet cells via tabletop centrifuge, aspirate supernatant, and lyse for 30 minutes on a rocker at 4 C (lysis buffer: 50 mM HEPES 7.4, 150 mM NaCl, 0.5% NP-40 detergent, 1 mM TCEP, cOmplete protease inhibitor tablet).
2. Pellet cells post lysis and recover supernatant
3. Measure protein content via a BCA assay. Briefly, reconstitute BCA detection reagent, dispense ~100 uL into each well of a 96 well plate, and prepare BSA stock solutions at 1, 2, and 3 mg/mL diluted in PBS. 

Set up a standard curve using the BSA samples, as well as blanks for lysis buffer and PBS. To prepare samples, add 2 uL of sample to the prepared 100 uL of BCA detection reagent.

Incubate sample at 37 C for 30 minutes, then scan on a plate reader using a BCA detection protocol. Calculate standard curve, and use to determine protein concentration in each sample. Check that BSA standard curve is linear, and that analyzed samples fall within the standard curve linear range.
4. Load 20 µg of samples in each lane of an SDS-PAGE, and run at 120 V for about 1.5 H. Run a prestained ladder
5. Recover gels and incubate in transfer buffer (48 mM Tris Base, 39 mM glycine, 0.037% SDS, 20% methanol) for 5 minutes. Also incubate a nitrocellulose sheet in transfer buffer.
6. Set up TransBlot apparatus, keeping transfer sandwich moist in transfer buffer. Transfer using standard protocol for 1 gel. 
7. Post transfer, use MW markers to cut strip at expected MW
8. Incubate strips in 5% (m/v) powdered milk in TBST at RT for 1 H or at 4 C while rocking to block.
9. Dilute primary antibodies according to manufacturer instructions in blocker buffer, incubate at RT for 1 H in a rocker.
10. Wash strips 3 x 5 min in TBST
11. Dilute secondary antibodies conjugated to HRP according to manufacturer instructions in blocking buffer, incubate at RT for 1 H in a rocker.
12. Wash strips 3x5 min in TBST.
13. Pick up strips and dry corner using kimwipe. Lays strips on saran wrap when excess buffer has been removed.
14. Reconstitute ECL substrate and pippete onto strips. Incubate for 5 min before dabbing off excess substrate and imaging using a chemiluminescent protocol on a chemidoc.
TITLE: Diagnosis of hypertension based on TCM (Traditional Chinese Medicine) constitution and wrist pulse wave signal
AUTHORS:  Lin Fan, Zhong-Ming Wang, Rong zhang, Yan Li, Xiao-Kang Zhang, Anonymous  

[DESCRIPTION]
In previous studies, pulse signals have been analysed primarily to standardise and objectify pulse diagnosis, but the influence of TCM constitution on pulse signals and disease has been neglected. Our study links the wrist pulse wave signal to TCM constitution to find TCM constitution factors that predispose to disease while improving the accuracy of disease classification. Our specific approach is set out below. A wrist pulse signal sampler based on a piezoelectric sensor was designed and 95 healthy subjects and 20 hypertensive patients were invited to complete the CCMQ (Constitution in the Chinese Medicine Questionnaire), of which 48 completed the pulse wave signal. Meanwhile, we improved the Butterworth filtering algorithm to denoise the pulse wave signals, and after smoothing and period segmentation, we finally extracted 27 time-domain features and 8 wavelet packet energy features. Then, we used the independent sample t-test and binary logistic regression analysis to analyze the correlation between the prevalence of hypertension and TCM constitution and found that the three constitutions of YaD (Yang-deficiency), YiD (Yin-deficiency), and PW (Phlegm-wet) were significantly associated with whether the subjects suffered from hypertension. The final classification results showed that the fusion features after adding the three constitution features with a high correlation with hypertension had a 17.46% higher F-score than the classification model without the constitution features. It can be seen that the incorporation of constitution characteristics into pulse characteristics in this paper has important implications for disease classification. This also provides a basis for studying the correlation between TCM physical characteristics and certain diseases, and patients can adopt targeted TCM management techniques to prevent diseases in advance.

[STEPS]
1. Pulse sampler

1.2. Design of pulse sampler 
1.1. Proposed pulse preprocessing method 
Pulse de-noising 
Period segmentation
1.3. Pulse signal feature extract 
Time-domain features 
Energy features of wavelet packet 
1.4. Statistical analysis 
Test of normality. 
Linearity test. 
Independent sample t-test. 
Binary Logistic Regression. 
2. Disease classification
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol to prepare Primer ID MiSeq sequencing library. Viral RNA was first extracted using QIAamp viral RNA extraction kit.  The block of random nucleotides (Ns) in the cDNA primers served as the Primer ID. The Superscript III kit was used for the cDNA synthesis. We used two rounds of PCR to amplify the cDNA and incorporate Illumina indexed adapters with KAPA2G Robust and KAPA HiFi PCR kits, respectively. 

[GUIDELINES]
<p><strong>Viral RNA extraction using QIAamp vRNA mini kit. </strong></p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/mqmfw26.png" width="591" height="443" data-src="https://s3.amazonaws.com/pr-journal/mqmfw26.png" data-ofn="PrimerID.png" /></strong></p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/mqpfw26.png" width="480" height="36" data-src="https://s3.amazonaws.com/pr-journal/mqnfw26.png" data-ofn="Screen Shot 2017-08-08 at 11.46.17 AM.png" /></strong></p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/mqrfw26.png" width="584" height="167" data-src="https://s3.amazonaws.com/pr-journal/mqqfw26.png" data-ofn="Screen Shot 2017-08-08 at 11.46.26 AM.png" /></strong></p>
<p><strong>Note: Primer ID primer and forward primer use random bases. Indexed primers have 24 fixed barcodes. </strong></p>
<p> </p>
<p><strong>Table of Indexed Primers</strong></p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/mqtfw26.png" width="739" height="378" data-src="https://s3.amazonaws.com/pr-journal/mqsfw26.png" data-ofn="Screen Shot 2017-08-08 at 11.48.36 AM.png" /></strong></p>
<p> </p>
<p> </p>

[STEPS]
SECTION: Prepare Primer Mix (Optional, only for multiplexed Primer ID library prep)
?. 
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SECTION: cDNA synthesis
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SECTION: Purification
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SECTION: PCR 1
?. 
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SECTION: Purification PCR products
?. 
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SECTION: PCR 2
?. 
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SECTION: Gel Purification and quantification
?. 
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TITLE: 2-step PCR mixture and conditions (Barcoded-head primers for seqs pooling)
AUTHORS: Yin-Tse Huang, Tsu-Chun Hung

[DESCRIPTION]
PCR mixture and condition (PowerPol 2X PCR Mix)

[STEPS]
1. Wear glove, clean up the working bench w. 1% bleach
SECTION: For 1&#x27; PCR head-primers
2. Prepare 1' PCR master mixutre for head-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for head-primers for each reaction
 

 

SECTION: For 1&#x27; PCR head-primers
3. Mix the 1' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 1&#x27; PCR head-primers
4. Transfer  23.75 µL 1' PCR master mixutre in 8-strip PCR tubes.

SECTION: For 1&#x27; PCR head-primers
5. Add 1.25 µLDNA template in 8-strip PCR tubes, resulting in a 25 µL reaction mixture for 1' PCR.


 

SECTION: For 1&#x27; PCR head-primers
6. Mix the reaction mixture gently by tapping the tubes. Quick spin the tubes.
SECTION: For 1&#x27; PCR head-primers
7. Carry out PCR using the following condition:  

1' PCR condition for head-primers
 

SECTION: For 1&#x27; PCR head-primers
8. Carry out electrophoresis for inspection of DNA products

   

SECTION: For 1&#x27; PCR head-primers
9. Markdown wells and upload the pictures to the Lab Google drive

  

SECTION: For 2&#x27; PCR barcoded-head primers
10. Prepare 2' PCR master mixutre for barcoded-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for barcoded-primers for each reaction (NO PRIMERs at this point!!)
 

 

SECTION: For 2&#x27; PCR barcoded-head primers
11. Mix the 2' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 2&#x27; PCR barcoded-head primers
12. Transfer 13.05 µL of the 2' PCR master mixture to 8-strip PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
13. Add 1.2 µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
14. Add 0.75 µL of 1' PCR product as template, resulting in 15 µL reaction mixture for 2' PCR.

Negative control contains only 14.25 µL master mixture and premixed barcoded-head primers but not DNA template
SECTION: For 2&#x27; PCR barcoded-head primers
15. Mix gently by tapping the tubes. Quick spin the tubes.
SECTION: For 2&#x27; PCR barcoded-head primers
16. Carry out 2' PCR using the following condition:  

2' PCR condition for barcoded-head primers
 

SECTION: For 2&#x27; PCR barcoded-head primers
17. Carry out electrophoresis for inspection of DNA products

  

SECTION: For 2&#x27; PCR barcoded-head primers
18. Markdown wells and upload the pictures to the Lab Google drive
TITLE: Anxa1 Immunostaining
AUTHORS: connor.davidson

[DESCRIPTION]
Protocol for perfusion, slicing, and immunostaining for Anxa1 in the striatum.

[STEPS]
SECTION: Perfusion
1. Anesthetize mouse in an anesthesia chamber set to 5% isoflurane, until breathing slows and foot pinch reflex stops.
SECTION: Perfusion
2. Restrain the anesthetized mouse with tape such that the chest is facing upwards, and all limbs are secured.
SECTION: Perfusion
3. Using surgical scissors, cut the skin away to reveal the ribs, and cut through the diaphragm and sides of the rib cage to expose the heart and lungs.
SECTION: Perfusion
4. Using a Masterflex L/S Easy-Flow peristaltic pump, insert the PBS needle into the left ventricle aimed at the left atrium, and secure the needle in place with forceps. 
SECTION: Perfusion
5. Cut right atrium with surgical scissors, and begin flow of 1x PBS at the max flow rate until the liquid from the chest cavity flows clear.
SECTION: Perfusion
6. Switch the source from PBS to 4% PFA, and maintain flow until mouse muscle reflexes from fixation ceases.
SECTION: Perfusion
7. Remove flow needle and sever head with thick surgical scissors.
SECTION: Perfusion
8. Using fine surgical scissors, remove scalp and cut the dorsal skull posterior to anterior, and pull skull away revealing fixed brain.
SECTION: Perfusion
9. Remove brain and store in 4% PFA for 24 hours.
SECTION: Perfusion
10. Transfer brain from 4% PFA to 30% sucrose solution for at least 2 days, or until the brain sinks to the bottom of the sucrose solution.
SECTION: Slicing
11. Place dry ice on all sides of the microtome, and wait until frost forms on the sides.
SECTION: Slicing
12. Create a small frozen 30% sucrose solution platform for the brain to rest on and freeze to
SECTION: Slicing
13. Cut cerebellum to create a flat surface on the brain so that the brain can rest on the platform with the olfactory bulbs facing upwards.
SECTION: Slicing
14. Wait until the brain has fully frozen before slicing coronal slices 50 microns thick, capturing the striatum with anterior posterior coordinates off bregma from 1.7 mm to -.34 mm.
SECTION: Slicing
15. Store coronal slices in 1x PBS
SECTION: Immunostaining
16. (All steps at room temp) Wash slices in 1x PBS 3 times, transferring from old PBS to new and letting sit for 5 minutes.
SECTION: Immunostaining
17. Soak slices in block solution (PBS 1x + .3% Triton-X + 5% Normal Goat Serum) for 30 minutes.
SECTION: Immunostaining
18. Transfer to block solution with Rabbit-anti-Anxa1 (Invitrogen #71-3400) primary antibodies (1:1000) for 1.5 hours, covered from light and on a rocking platform.
SECTION: Immunostaining
19. Wash in 1x PBS + 0.3% Triton-X 4 times for 5 minutes each.
SECTION: Immunostaining
20. Transfer to block solution with goat-anti rabbit (Invitrogen #A11037) antibodies (1:250) for 2 hours, covered and rocking.
SECTION: Immunostaining
21. Wash in 1x PBS 3 times for 5 minutes each, and store slices in PBS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In order to increase the number of viral particles a blind passage of sera might be performed in <em>Aedes albopictus C6/36</em> cells.</p>

[STEPS]
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TITLE: Parallel Beam test for mice
AUTHORS: Cristian González-Cabrera, Katharina Draggendorf, Matthias Prigge

[DESCRIPTION]
The Parallel Beam Test Protocol is designed to evaluate the motor coordination of mice. The test involves a 150 cm long balance beam set at a height of 50-60 cm, with safety measures to catch falls. Conducted in a dimly lit environment with focused lighting on the beam, the test requires mice of any strain to traverse the beam, guided initially towards their home cage. Mice are trained a few days before testing and acclimatized to the test environment an hour prior to minimize stress. Each mouse completes three consecutive attempts, recorded for analysis. Post-run, the beam is cleaned with 10% ethanol. This protocol is essential for assessing motor skills, particularly in neurological disease such as Parkinson Disease

[STEPS]
SECTION: Objective
1. This protocol allows for the evaluation of balance and coordination in mice, which can be crucial for studies involving neurological conditions, muscular diseases, or the effects of drugs on motor functions
SECTION: Apparatus
2. A 150 cm long balance beam placed on two opposing supports.
The height between the beam and the ground should be 50-60 cm, with a safety net or cushioning material to catch any falls.
A side-mounted camera to record the test.

SECTION: Apparatus
3. Lighting

The environment should be dark, with a bright light directed onto the beam.

SECTION: Preparation
4. Acclimate mice to the test environment and the handler before the test.
A few days before the first test run, train the mice on the beam. Place the mouse on one end of the beam and guide it towards its home cage at the other end, encouraging it to walk across the beam.
Repeat this process 3-5 times until the mouse can traverse from the start to the opposite platform without the home cage as an incentive.
SECTION: Acclimatization:
5. Bring mice in their home cage to the behavioral room at least 1 hour before testing to minimize stress
SECTION: Test Procedure
6. Each mouse completes three consecutive runs on the beam, all recorded by the camera.
Clean the beam with 10% ethanol after each mouse to maintain hygiene and consistent conditions
SECTION: Test Procedure
7. Additional Considerations:

The beam should have a uniform surface without any textures or protrusions that might aid in grip.
The performance can be quantified by measuring the time it takes for the mouse to traverse the beam, the number of slips or falls, and the overall gait pattern.
It's important to ensure that the mice are not injured during falls. The safety net or cushioning should be appropriately placed and tested.
The data from the camera recordings can be analyzed for various parameters like speed, balance, and hesitation instances to provide a comprehensive assessment of motor coordination.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a very handy, previously published [Ref 1 and 2], rhinovirus (RV) screening and genotyping assay which I and many others have used on many sample extracts, mostly originating from acutely ill paediatric patients, spanning well over a decade's worth of collection dates.</p>
<p> </p>
<p>I have not confirmed that it can detect every single RV genotype but I do know that it detects <em>many</em> from each of the three RV species (<em>Human rhinovirus A</em>, <em>Human rhinovirus B</em> and <em>Human rhinovirus C</em>) as well as at least some <em>Human enterovirus</em> (EV) genotypes.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/u6edyxe.png" data-src="https://s3.amazonaws.com/pr-journal/u6edyxe.png" data-ofn="RV-5" /></p>
<p> </p>
<p>The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region.</p>
<p> </p>
<p>This assay produces a useful backup subgenomic sequencing target for use primarily should the 'Wisdom VP42' protocol fail to amplify a product for sequencing. It is worth noting that these primers span a target region that can produce confusing results. Recombination within the 5' end of the genome of some members of the genus <em>Enterovirus</em> can lead to 5'UTR sequences generated by RV-C genotypes that appear to be RV-As (see the figure below in which green RV-Cs appear in what is expected to be the red RV-A branch of the phylogenetic tree). </p>
<p> </p>
<p> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/vdfdyxe.png" data-src="https://s3.amazonaws.com/pr-journal/vdedyxe.png" data-ofn="Recombination_UTR Trees vs polyprotein.png" /></p>

[STEPS]
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TITLE: MojoSort™ Human Pan DC Isolation Kit Protocol
AUTHORS: Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol covers usage of BioLegend's MojoSort™ Human Pan DC Isolation Kit Protocol.</div></div>

[STEPS]
?. Prepare cells from your tissue of interest or blood without lysing erythrocytes.
?. In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4 mL in a 5 mL (12 x 75 mm) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. Filter the cells with a 70 µm cell strainer, centrifuge at 300 x g for 5 minutes, and resuspend in an appropriate volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108cells/mL.
?. Aliquot 100 µL of cell suspension (107cells) into a new tube. Add 10 µL of Human TruStain FcX™. Mix well and incubate at room temperature for 10 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of Human TruStain FcX™ for 1x107cells is 10 µL, add 100 µL for 1 x 108cells. When working with less than 107cells, use indicated volumes for 107cells.
?. Add 10 µL of the Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of Antibody Cocktail for separating 1 x 108cells in 1 mL of MojoSort™ Buffer. When working with less than 107cells, use indicated volumes for 107cells.Optional: Take an aliquot before adding the cocktail to monitor purity and yield.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 20 µL of Streptavidin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 200 µL of Nanobeads for separating 1 x 108cells in 1 mL of MojoSort™ Buffer. When working with less than 107cells, use indicated volumes for 107cells.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300 x g for 5 minutes.
?. Discard supernatant.
?. Add 2.5 mL of MojoSort™ Buffer.Note: If you observe aggregates, filter the suspension. To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
?. Place the tube in the magnet for 5 minutes.
Optional: Take a small aliquot before placing the tube in the magnet to monitor purity and yield. Keep unused cells to be used as control or other applications if needed.
?. Pour out and collect the liquid. These are your cells of interest; DO NOT DISCARD. Resuspend the labeled cells in 2.5 mL MojoSort™ Buffer.