eamag/protocols-with-steps huggingface dataset:
TITLE: Determining biofilm growth amount (absorbance) 
AUTHORS: An.Huang

[DESCRIPTION]
This protocol describes a method to determine the growth amount of biofilm at the early stage of biofilm formation by measuring absorbance. Here we use our own engineered bacteria, and it requires induction of IPTG and cultured with silver ion. You may use a different kind of bacteria and different culturing medium.

[STEPS]
SECTION: IPTG induction
1. 
Escherichia coli grown overnight was diluted by LB to OD600=0.4-0.6.
SECTION: IPTG induction
2. IPTG was added to cell culture to 1mM IPTG finally, and incubated 3h at 171 rpm, 37℃ in orbital shaking incubator.
SECTION: Sample preparation
3. Preparing several flasks by filling the flasks with MBBR carrier K1. Autoclave all the flasks and dry them in an oven at 60s℃.
 


SECTION: Sample preparation
4. Cells cultures and silver nitrate (to 6μM) solution were mixed in advance. Add the mixture into each bottle.
 

SECTION: Sample preparation
5. Incubate all bottles in a biochemical incubator at37 °C .

SECTION: Absorbance measurement
6. At the day of measuring the absorbance, take the flasks to be tested out from incubator.
SECTION: Absorbance measurement
7. Using a pair of forceps, carefully pick out the carriers that has not been submerged into the culture and discard.
 

SECTION: Absorbance measurement
8. Randomly pick five carriers out from culturing flask and put into a 50 mL centrifuge tube. Label this tube as "Washing tube".
 

SECTION: Absorbance measurement
9. Wash these five carriers using 1X phosphate-buffered saline (PBS) for three times. Collect all eluents.
 

SECTION: Absorbance measurement
10. Use a dropper, add 2-3 mL 1X PBS into the Washing tube. Cap the tube and rinse every corner of the inside wall of tube using the PBS added by rotating the tube. Collecting the eluents by pouring these PBS into Eluent tube. Repeat these steps for three to five times.

 

SECTION: Absorbance measurement
11. Centrifuge the Eluent tube at10000 rpm, 10 min .

SECTION: Absorbance measurement
12. Discard all supernatants.

 

SECTION: Absorbance measurement
13. Add 3 µL  1X PBS into Eluent tube. Resuspend the pellet.

SECTION: Absorbance measurement
14. Measure the absorbance of the liquid   at OD600. Record the absorbance.

SECTION: Absorbance measurement
15. Repeat steps from Step#7 to Step#14 for three times. Calculate the mean value for the three measurements. A growing curve of biofilm can be generated from the data collected.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Reagent List:</strong></p>
<ul>
<li>Sterile PBS</li>
<li>Cell culture medium (IMDM supplemented with 10% FBS)</li>
<li>Sterile plastic petri dishes</li>
<li>RBC Lysis Buffer (Cat. No. 420301)</li>
<li>Anti-mouse CD3ε, clone 145-2C11 (LEAF™ format, Cat. No. 100314)</li>
<li>Anti-mouse CD28, clone 37.51, (LEAF™ format, Cat. No. 102112)</li>
<li>Anti-mouse IFN-γ, clone XMG1.2, (LEAF™ format, Cat. No. 505812)</li>
<li>Anti-mouse IL-4, clone 11B11, (LEAF™ format, Cat. No. 504108)</li>
<li>Recombinant mouse IL-6 (carrier-free) (Cat. No. 575704)</li>
<li>Recombinant mouse IL-23 (carrier-free) (Cat. No. 589002)</li>
<li>Recombinant human TGF-β1 (carrier-free) (Cat. No. 580702)</li>
<li>Brefeldin A (Cat. No. 420601)</li>
<li>Monensin Solution (Cat. No. 420701)</li>
<li>PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)</li>
<li>Ionomycin (Cat. No. I0634 from Sigma)</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Preparing Borax RapidBuffer for electrophoresis
AUTHORS: Jenny  Molloy, Nadine Mowoh, Stephane Fadanka, Cordellia Fulai

[DESCRIPTION]
Molecular biologists often use tris-based conduction buffers like TBE or TAE in running DNA gels. These buffers are known to overheat at high voltages, causing problems with gel integrity, sample denaturation and more. The voltage thus needs to be limited to a maximum of 5-10 volts/cm (e.g 100 volts for a 10 cm gel) leading to extended running time, sometimes to hours.

With 10mM Sodium Tetraborate (CAS#: 1330-43-4, Na2B4O7, Borax), the voltage can be increased to 35 volts/cm without any problem, reducing the time taken to run gels by up to 7 times for standard applications (e.g. separation of DNA fragments from 100 bp - 5 kbp).

Our lab’s standard DNA gel buffer has become 20 g of Borax into 1 l of water which gives a 20x stock solution. Small gels run in 10-15 minutes at 200V.

 SCOPE: 

Preparation of gel electrophoresis buffer for separation of DNA fragments from 100 bp - 5 kbp




[STEPS]
SECTION: Preparation of 10x Borax RapidBuffer Solution
7. Pour the prepared solution into a clean, labelled screw-top 1000 mL glass or plastic storage bottle and store at Room temperature until use.

SECTION: Preparation of 10x Borax RapidBuffer Solution
6. Add distilled or de-ionised water to a total of 1000 mL and stir again for 3 min

SECTION: Preparation of 10x Borax RapidBuffer Solution
5. Add a magnetic flea and use a magnetic stirrer at Room temperature to fully dissolve the borax powder.

SECTION: Preparation of 10x Borax RapidBuffer Solution
4. Using a 1000 mL measuring cylinder, measure800 mL of distilled or deionised water and add to the 1000 mL beaker.
 
SECTION: Preparation of 10x Borax RapidBuffer Solution
3. Pour powder into a graduated 1000 mL glass beaker or flask

SECTION: Preparation of 10x Borax RapidBuffer Solution
2. Use a spatula to measure out and weigh 10 g of Borax (Sodium Tetraborate, CAS#: 1330-43-4)
SECTION: Preparation of 1x Borax RapidBuffer Solution (500ml)
10. 1X RapidBuffer solution is now ready for use and can be stored at Room temperature for use within one month.

SECTION: Preparation of 1x Borax RapidBuffer Solution (500ml)
9. Add approx 450 mL distilled or deionised water, sufficient to make up the volume to 500 mL

SECTION: Preparation of 1x Borax RapidBuffer Solution (500ml)
8. Use a 100 mL measuring cylinder to measure out 50 mLof 10X RapidBuffer into a 500 mL graduated beaker or flask.

SECTION: Preparing
1. Weigh out a certain amount of borate and place into a vacuum Tupperware half filled with silica beads.
place the lid on top and cover tightly, then use the vacuum pump to extract air inside and allow to dry for720 min at 37 °C).



1.2 Carefully pour it out into zip lock bags and seal to avoid it from absorbing moisture and store in cool dry place.
TITLE: Recipe for 50x TAE buffer
AUTHORS: Anna Behle, Alice Pawlowski

[DESCRIPTION]
Stock solution for 50x TAE. TAE buffer is a solution made up of Tris base, acetic acid and EDTA (Tris-acetate-EDTA). It is a common buffer for DNA separation using standard agarose gel electrophoresis.

[STEPS]
SECTION: Preparation of a 0.5 M EDTA stock solution
2. For 500 ml:
weigh out 93.05 grams of EDTA disodium salt (MW=372.24 g/mol)
Dissolve in 400 milliliter deionized water and adjust the pH with solid sodium hydroxide (NaOH) plates, EDTA will not go completely into solution until the pH is adjusted to about 8.0!
Top up the solution to a final volume of 500 milliliters
autoclave
SECTION: Preparation of 50 x TAE buffer
3. weigh out 242 grams of Tris-base (MW = 121.14 g/mol) and dissolve in approximately 700 milliliters of deionized water
Carefully add 57.1 milliliters of 100 % glacial acid (or acetic acid) and 100 milliliters of 0.5 M EDTA (pH 8.0)
adjust the solution to a final volume of 1 liter
the pH of this buffer is not adjusted and should be about 8.5
store stock solution at room temperature
SECTION: Preparation of 1 x TAE working solutuion
4.  
20 mL 50 x TAE
add 920 mL DI H2O
 
final concentration in gel / running buffer: 40 mM Tris, 20 mM acetic acid, 1 mM EDTA
SECTION: Components required
1.    Component Molarity in 50x Molecular weight     EDTA disodium salt 50 mM 372.24 g/mol     Tris 2 M  121.14 g/mol     glacial / acetic acid 1 M  60.05
TITLE: Open field test 
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
The open field test is a simple test to evaluate general motor and explorative behavior in rodents 

[STEPS]
2. Place the animal in the center of open field cylindrical arena (100 cm diameter) with dark walls (35 cm) and floor, under dim lighting (300 lux)
1. Habituate the animal for 1 h to the testing room before the test 
3. Leave the animal free to explore the arena for 10min and record  with a video camera suspended above the arena
4. Track the center and the periphery with video tracking software (EthoVision - Noldus, The Netherlands)
5. Clean the apparatus every time between animals
TITLE: Zymo OneStep PCR Inhibitor Removal Kit
AUTHORS: Shannon Fitz, Alex Shaw, michael Owusu, Dilip Abraham

[DESCRIPTION]
Features:
• For high quality DNA or RNA that is free of enzymatic inhibitors including polyphenolics, humic/fulvic acids, tannins, melanin, etc.
• Fast, one-step procedure for “cleaning” impure samples prior to PCR, sequencing, RT, etc.

Description:
The OneStep‱ PCR Inhibitor Removal Kit contains all the components needed to efficiently remove contaminants from DNA/RNA preparations that can inhibit downstream enzymatic reactions such as PCR and RT. The column matrix has been specifically designed for efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from most impure DNA and RNA preparations. Sample cleanup is as simple as: applying, spinning and recovering a sample from the column.

The steps and abstract for this protocol consist entirely of the manufacturer's instructions with no deviations, as per https://files.zymoresearch.com/protocols/_d6030_onestep_pcr_inhibitor_removal_kit.pdf. 

[STEPS]
SECTION: Column Preparation
1. Insert column into a Collection Tube. 

*Please note that matrix in the column may appear dehydrated, or powdery. This is normal.
SECTION: Column Preparation
2. Open the cap, add 600 µl of Prep-Solution and centrifuge at 8,000 x g for 3 minutes. 
SECTION: Column Preparation
3. Inhibitor Removal:

Transfer the prepared column to a clean 1.5 ml microcentrifuge tube. Add 50 - 200 µl DNA or RNA (in water, TE, or similar) to the Zymo-Spin‱ III-HRC Column and centrifuge at 16,000 x g for 3 minutes. 

The filtered DNA (or RNA) is suitable for PCR, (RT), and other downstream applications.
TITLE: Liposome tubulation
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the LRRK2-induced liposome tubulation experiment and its analysis by confocal fluorescence microscopy and negative stained electron microscopy.

[STEPS]
SECTION: Confocal fluorescence microscopy analysis
1. Prepare the samples in a PCR tube with 300 nanomolar (nM) LRKK2 proteins (WT or mutant full length LRRK2 or RCKW), 20 micromolar (µM) liposomes with or without 1 millimolar (mM) GMPPNP (or other guanylnucleotides).
 

SECTION: Confocal fluorescence microscopy analysis
2. Immediately deposit 6 µL -10 µL samples of step 1 on a   glass bottom dish and incubate at 37 °C for 30 min.
 

SECTION: Confocal fluorescence microscopy analysis
3. After incubation, capture  images  with a Spinning disk confocal (SDC) microscopy at Room temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system (Perkin-Elmer).
 

SECTION: Negative stained electron microscopy (EM) analysis
4. Glow-discharge carbon-coated grids (25 mA, 45 s).
SECTION: Negative stained electron microscopy (EM) analysis
5. Place the discharged grids into a   glass bottom dish.
SECTION: Negative stained electron microscopy (EM) analysis
6. Prepare samples in a PCR tube with 300 nanomolar (nM) LRKK2, 80 micromolar (µM) liposomes and 1 millimolar (mM) GMPPNP.
SECTION: Negative stained electron microscopy (EM) analysis
7. Immediately apply 6 µL of the mixture to the grid and incubate the mixture at 37 °C for 30 min.
 

SECTION: Negative stained electron microscopy (EM) analysis
8. Blot the grid with filter paper after incubation and stain samples with 2% uranyl acetate for 40 s.
SECTION: Negative stained electron microscopy (EM) analysis
9. Dry the grid with filter paper.
SECTION: Negative stained electron microscopy (EM) analysis
10. Take images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS Camera (Thermo Fisher Scientific).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Objective </strong></p>
<p>To determine whether the topical application of platelet-rich plasma (PRP) promotes healing in experimentally-induced full-thickness skin wounds in animals, a systematic review and meta-analysis will be performed.</p>
<p><strong>Methods</strong></p>
<p>Web of Science, Cochrane Library, PubMed, Research Gate, Cochrane Wounds Group, and Veterinary Information Network will be searched to identify randomised and not randomised controlled clinical trials comparing PRP with placebo or with other treatments in animals. Primary outcome: reduction of open wound area; secondary outcomes: healing time and number of healed cases. Effect sizes: Hedges’ g; odds ratio. </p>
<p> </p>
<p>This protocol was used in the following publication:</p>
<p><a href="https://doi.org/10.1371/journal.pone.0191093" target="_blank">https://doi.org/10.1371/journal.pone.0191093</a></p>
<p>Tambella AM, Attili AR, Dupré G, Cantalamessa A, Martin S, Cuteri V, Marcazzan S, Del Fabbro M (2018) Platelet-rich plasma to treat experimentally-induced skin wounds in animals: A systematic review and meta-analysis. PLoS ONE 13(1): e0191093. doi: <a href="https://dx.doi.org/10.1371/journal.pone.0191093" target="_blank">10.1371/journal.pone.0191093</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: UPitt TriState SenNet TMC FFPE Specimen Section 
AUTHORS: John Sembrat, Marta Bueno

[DESCRIPTION]
To perform imaging analysis of the FFPE blocks were submitted to hematoxylin and eosin stain. Initially FFPE blocks were prepare by the Research Histology Pitt Biospecimen Core and kept at room temperature (preserved from light) until sectioning.

[BEFORE_START]
Monitor the water bath temperature carefully. The temperature will need to be 5 ‑ 9 ˚C below the melting point of the wax. Make sure the water is clean and free of bubbles and section waste (to avoid cross‑contamination).
Make sure the appropriate slides are ready.

[GUIDELINES]
Sectioning is generally improved when the specimen and the wax are well matched in hardness. It is for this reason that most paraffin blocks must be cold
when sections are cut.
The choice of slide and adhesive will be influenced by the staining methods to be subsequently applied.
It is important to remove accumulated tissue debris and wax after use. Regular preventative maintenance is important

[STEPS]
SECTION: Section Collection
1. Using a Histo-Quill Pen, hand label all slides with project number and block ID (add type of stain if needed) .
SECTION: Section Collection
2. Ensure paraffin block is at room temperature.
SECTION: Section Collection
3. Place block into holder of microtome.
SECTION: Section Collection
4. Use clean, sharp microtome knife or disposable blade.
SECTION: Section Collection
5. Cut section to a 5 μm thickness.   

SECTION: Section Collection
6. Float paraffin ribbons on deionized H2O (or RNase-free H2O), heated to just below the melting point of the paraffin being used (usually 42 °C).

SECTION: Section Collection
7. Mount sections on slide by slowly bringing the flat side of slide up from underneath the floating tissue section, so that the tissue section is centered in the membrane window.
SECTION: Section Collection
8. Place slide at an angle to allow water to drain and incubate sample at room temperature for 90 min.

SECTION: Section Collection
9. Slides can now be stored at room temperature for future use.
SECTION: Section Collection
10. Proceed with staining protocol (if needed).
SECTION: Section Collection
4.1. Use forceps or brush instead of your fingers to pick up sections or wax fragments from blade or block face.
SECTION: Section Collection
4.2. The knife or blade should be removed from the microtome when the instrument is left unattended or when cleaning the instrument. It can then be grasped with forceps (not fingers) or picked up with the magnet at the end of a brush and safely removed. Used blades should be disposed of appropriately in a “sharps” container.
SECTION: Section Collection
4.3. Set blade clearance to the optimal clearance angle & orient the specimen in the appropriate position.
SECTION: Section Collection
5.1. Note: Dip block face into molten paraffin to seal the tissue and allow to cool before storage.
TITLE: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
AUTHORS: Patricia Yuste-Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently monitor α-Synuclein aggregation by thioflavin T fluorescence in a plate reader.

[STEPS]
SECTION: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
1. Mix reagents to a final concentration of 200 micromolar (µM) α-Synuclein, 0.05 % NaN3,
10 micromolar (µM) ThT in α-Synuclein fibril buffer. Prepare a mix for 4.5 reactions per condition (per condition, four technical replicates in each plate). Final volume is 80 µL per well. 
 

SECTION: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
2. Dispense 80 µL of the mix into a well of a 96 well half-area plate of black polystyrene with a clear bottom.

SECTION: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
3. If possible, the outer wells should not be used and should be filled them with water.
SECTION: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
4. Seal the plate with parafilm to avoid evaporation.
SECTION: α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
5. Set the following parameters in a SPARK multimode microplate reader (TECAN) and start the reaction:
Fluorescence measurement: ThT signal, excitation 440 nm, emission 480 nm, (use gain regulation) measured every 10 min.
Temperature 37 °C
Constant shaking (2 min linear shaking: amplitude 1.5 mm, frequency 1080 rpm - 2 min orbital shaking: amplitude 1 mm, frequency 510 rpm).
TITLE: Immunohistochemistry Protocol for Keratin Antibodies
AUTHORS: Sam Li

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

[STEPS]
?. [Clear Slides: Removes paraffin and hydrates the tissue]
Note:If using frozen sections, allow slides to come to room temperature for 15 minutes & proceed to step (F) only A. Xylene:
5 minutes in each of (3) different 250mL containersB. 100% alcohol
5 minutes in each of (3) different 250mL containersC. 95% alcohol
3 minutes in (1) 250mL containerD. 70% alcohol
3 minutes in (1) 250mL containerE. Water
1 minutes in each of (3) different 250mL containersF. H2O2 (3%)
15 minutes in (1) 250mL container
?. [Rinse Slides]
Rinse slides with lab grade water.Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionisation (RODI).
?. [Antigen Retrieval]
A. 70% Formic Acid - incubate the slides for 20 minutes at room temperature. Note: This antigen retrieval step is harsh on the tissue. If using frozen sections reduce time to 5-10 minutes or omit if tissue falls off the slide.B. Rinse Slides with 1X PBS.C. Remove from microwave and allow slides to cool on the bench top for 10 minutes.D. Rinse slides with lab grade water.
?. Apply serum block for at least 5 minutes.Do not wash after this step.
?. Blot off serum block
?. Apply primary antibody (see recommended dilution from datasheet).
?. Incubate primary antibody 60 minutes at room temperature.
?. Rinse slides with 1X PBS.
?. Apply USA Linking reagent - 20 minutes incubation.
?. Rinse slides with 1X PBS.
?. Apply Labeling Reagent - 20 minutes incubation
?. Rinse with 1X PBS.
?. Apply chromogen - 5 minutes incubation. Dilute according to manufacturer's instructions.A. AEC Chromogen: 20µL AEC chromogen + 1mL AEC substrate buffer
?. Rinse slides with lab grade water.
?. [Coverslip]
A. Submerge slides in Mayer’s Hematoxylin for 30 secondsB. Rinse under running lab grade water for 1 minute or until water is clear.C. Submerge slides in Bluing Reagent for 1 minute.D. Rinse under running lab grade water for 1 minute.E. Cover slip slide using Permanent Aqueous Mounting Medium.Note: do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.
TITLE: ND5 sequencing of Oncorhynchus masou masou
AUTHORS: Yoko Kato-Unoki

[STEPS]
?. Amplify the ND5 region (1597 bp) with the following PCR mixture and program.  AB1ComponentAmount25× Phusion HF buffer (New England BioLabs)2 μl3Phusion DNA Polymerase (New England BioLabs)0.1 μl410 mM dNTPs0.2 μl510 μM primer ND5-F10.5 μl610 μM primer ND5-R0.5 μl7Genomic DNA10–50 ng8Nuclease-free waterVariable9Total volume10 μl
AB1ComponentAmount25× Phusion HF buffer (New England BioLabs)2 μl3Phusion DNA Polymerase (New England BioLabs)0.1 μl410 mM dNTPs0.2 μl510 μM primer ND5-F10.5 μl610 μM primer ND5-R0.5 μl7Genomic DNA10–50 ng8Nuclease-free waterVariable9Total volume10 μl
?. Electrophorese 1 μl PCR product through a 1% agarose–TAE gel.Check for the presence of non-specific amplification products and/or primer-dimers, and calculate the approximate concentration of the specific amplified DNA fragment by comparing its band intensity to that of the marker.
?. Sequence the PCR product with the primers ND5-F1 or ND5-F2.Note: The PCR product can be used directly as a sequence template without purification or clean-up reagents if there is no non-specific amplification, no primer-dimer contamination, and can be diluted more than 5-fold in the sequence reaction solution.
?. Assemble and align the ND5 sequences by using ATGC Ver. 4.3.5 software (GENETYX Co.).
TITLE: Example PLSR for Predicting Leaf Traits from Leaf Spectra
AUTHORS: Angela C Burnett, Jeremiah Anderson, Kenneth J Davidson, Kim S Ely, Julien Lamour, Qianyu Li, Bailey Morrison, Dedi Yang, Alistair Rogers, Shawn P Serbin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for predicting leaf traits from leaf reflectance spectra, using partial least-squares regression (PLSR).</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> </p>
<p>Embodied cognition research suggests that bodily experiences might ground mental representations of emotional valence in the vertical dimension of space (i.e., positive is up and negative is down). Accordingly, recent studies show that upward and downward arm movements may also influence the evaluation of valence-laden stimuli, suggesting that upward (downwards) movements lead to more positive (negative) evaluations. Interestingly, these studies typically did not investigate paradigms that require a direct hand interaction with the stimuli. With the advent of touchscreen devices and their use for experimental environments, however, a direct and more natural hand interaction with the stimuli has come to the fore. In this regard, the goal of the present study is to examine how direct hand interaction with valence-laden stimuli on a touchscreen monitor affects their perceived valence. To do so, participants evaluated emotional pictures after touching and moving them either upwards or downwards across a vertically mounted touchscreen. In contrast to previous findings, the results suggest that positive pictures were evaluated as more positive after downward movements while negative pictures were evaluated as less negative following upward movements. This finding may indicate that a matching between the pictures’ valence and the valence associated with their vertical touch location leads to more positive evaluations. Thus, the present study extends earlier results by an important point: Touching the emotional pictures during movement may influence their valence processing.</p>

[STEPS]
?. 
?. 
?.