eamag/protocols-with-steps huggingface dataset:
TITLE: Preparation of Glycerol Stocks from storing microbes from Petri Dishes
AUTHORS: Stephane Fadanka, Shalo Minette, Nadine Mowoh

[DESCRIPTION]
This protocol is meant to provide researchers with a step by step procedure on how to prepare glycerol stocks in order to preserve and store bacteria for long term. 
Bacterial glycerol stocks are important for long-term storage of plasmids. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years and -20°C for several months.

Glycerol reduces the harmful effects of ice crystals on bacteria which cause dehydration and damage cells through a localised increase in salt concentration leading to the denaturation of proteins. 


[BEFORE_START]
Make sure all materials and reagents needed for this protocol are available

[GUIDELINES]
This protocol describes the steps in preparing bacterial glycerol stocks. This protocol can be performed by anyone with basic molecular biology skills.

[STEPS]
SECTION: From the plate to a PBS eppendorf
3. If you have colonies already growing in a Petri dish you can simply collect them using an inoculating wire loop and transfer to a eppendorf tube with  500 µL  of 1x PBS
Once you have collect enough pure colonies you can go to the next step.
SECTION: Diluting Pure Glycerol to 50% with Distilled water
1. Use a clean measuring cylinder to measure 10 mLof distilled water and equal amount 10 mL of   into a 50 mL falcon tube.

Cork the tube and shake thoroughly until the liquids are evenly mixed

SECTION: Filtering the 50% glycerol
2. Use a 20 mL sterile syringe to aspirate the 50% glycerol from the falcon tube 

Plug in a 0.2 µM micro filter and filter out the glycerol into a sterile falcon tube


SECTION: Aliquoting the bacterial culture into 50% glycerol and storing
4. To make 1mL of Bacteria glycerol stocks, aliquot 500 µLof the filtered 50% glycerol into separate 1.5 mL Eppendorf tubes (triplicates or more depending on the quantity of glycerol stocks needed.
Use sterile micropipette and tips to measure out equal volumes (500 µL) of the bacterial culture from the Erlenmeyer flask into the tubes containing the 50% glycerol (We now have equal proportion of bacteria and 50% glycerol).

Keep your thumb pressed firmly against the lid of the Eppendorf tube and  shake vigorously to make sure the 2 liquids mix completely.

Use a marker pen to label the tubes with the name of the bacterial strain and date of preparation of the glycerol stock

Store the vials in the freezer at -20°C until they are used.
TITLE: Detection of anti-phytopathogenic fungal activity
AUTHORS: Cecilia Monica Rodriguez García, Leticia Peraza Echeverría

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe the detection of in vitro anti-phytopathogenic fungal activity using the broth dilution method to find the MIC (minimum inhibitory concentration) of an aqueous extract of leaves. Four concentrations were assayed: 1, 2.5, 5, and 10% (v/v) in PDB medium. Each concentration was inoculated with a spore suspension of the selected fungus. PDB without spores was used as negative control and with the addition of a commercial fungicide like positive control. After the inoculation of the fungus, the tubes were incubated for three days in the dark at 26-28 °C. After the incubation time, the tubes were checked for growth of the fungus, if no growth is observed, 25 μL of the suspension are transferred to PDA medium in Petri dishes of 3 cm diameter. Evaluations were made on day 7. The MIC was determined as the lowest concentration of aqueous extract preventing the growth of macroscopically visible mycelium.</div></div>

[STEPS]
?. [Dilution of the aqueous extract of leaves ]
ABCDEF1Dilution numberAE (μL)CS&nbsp;
(μL)
H2O
(μL)
&nbsp;final volume
(μL)
concentration
(%)
21&nbsp;500&nbsp;100&nbsp;400&nbsp;1000532&nbsp;250&nbsp;100&nbsp;650&nbsp;10002.543&nbsp;125&nbsp;100&nbsp;775&nbsp;10001.255&nbsp;&nbsp;---&nbsp;---&nbsp;---&nbsp;100010 CS- conidial suspension (200 conidium/μL)
ABCDEF1Dilution numberAE (μL)CS&nbsp;
(μL)
H2O
(μL)
&nbsp;final volume
(μL)
concentration
(%)
21&nbsp;500&nbsp;100&nbsp;400&nbsp;1000532&nbsp;250&nbsp;100&nbsp;650&nbsp;10002.543&nbsp;125&nbsp;100&nbsp;775&nbsp;10001.255&nbsp;&nbsp;---&nbsp;---&nbsp;---&nbsp;100010
Do not add de CS, only make the mix of AE and water, CS is added tube by tube
?. [Dilution of the aqueous extract of leaves]
Add to a sterile 1.5 mL eppendorf tube mix 1 and then 12.5 μL of CS (in duplicate)Repeat num 1 for the next concentratión and so on..For the 10% AE just put in each tube 125 μL 10% AE plus 12.5 μL of CS, (in duplicate)The positive control is prepared by adding 1 μL of a commercial fungicide to 125 μL of water and 12.5 μL of CS (in duplicate)Two negative controls: one  is only 125 μL of water and the other is 125 μL of PDB and both with 12.5 μL of CS (in duplicate)
?. [Bioassay conditions]
Once the conidial suspension has been added, homogenize very well, seal the tubes and leave them in the dark at a temperature of 26-28 ° C for 3 days.
?. [Bioassay conditions]
After three days, 25 μL of the suspension is transferred in the center of the PDA medium (polystyrene dish, Corning), in duplicate.
The suspension of each eppendorf tube must be perfectly homogenized
?. [Bioassay conditions]
Petri dishes are incubated at 26 ± 2 °C at 12/12 photoperiod for 7 days (two replicates /concentration)Evaluation are made on day 7
If the aqueous extract of leaves is fungicidal there should be no growth on the PDA medium, if the extract is not fungicidal then there must be growth of the fungus on the PDA medium
TITLE: Expression and purification protocol of linear GST-4xUbiquitin
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the expression and purification of linear GST-4x Ubiquitin.</div></div>

[STEPS]
?. [Protein purification]
Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at  at  for
Centrifuge: 16000 33
4 °C
?. [Protein purification]
Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at  with gentle shaking for , apply to a gravity flow column, and wash extensively with Wash Buffer.
4 °C
?. [Protein purification]
Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer operating at .
4 °C
?. [Protein purification]
Pool the peak fractions containing pure protein, store the snap-frozen in liquid nitrogen at .
-80 °C
?. [Protein expression]
Transform the E.Coli BL21DE3 cells with a plasmid encoding for GST-tetraubiquitin and plate them on Amp plate.
?. [Protein expression]
Carry out the protein expression in  LB meidum, induce with  IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at .
1.5 L
18 °C
?. [Protein expression]
Harvest the cells by spinning at  for  at  and stock at  until purification.
Centrifuge: 4500 34
4 °C
-80 °C
?. [Protein purification]
Follow the GST batch purification by Size Exclusion Chromatography.
TITLE: Teacher Sense of Efficacy Scale Short Form
AUTHORS: Kevin Crouse

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Teachers’ Sense  of Efficacy is the beliefs in their capability to make a difference in  student learning, to be able to get through even to students who are  difficult or unmotivated</div><div class = "text-block">d. The Teacher Sense of Efficacy Scale asks  teachers to assess their capability concerning instructional strategies,  student engagement, and classroom management.</div><div class = "text-block"><span>This protocol taken from </span><span style = "font-weight:bold;">Megan Tschannen-Moran'</span><span>s classic work on </span><span style = "font-style:italic;">Teacher Self-Efficacy</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. How much can you do to control disruptive behavior in the classroom?
?. How much can you do to motivate students who show low interest in school work?
212 °C
?. How much can you do to calm a student who is disruptive or noisy?BUG (for social science use): GOTO does not allow you to skip, only to go back.  Also, no highlighting text.
?. How much can you do to help your students value learning?
?. To what extent can you craft good questions for your students?
?. How much can you do to get children to follow classroom rules?
?. How much can you do to get students to believe they can do well in school work?
?. How well can you establish a classroom management system with each group of students?
?. To what extent can you use a variety of assessment strategies?
?. To what extent can you provide an alternative explanation or example when students are confused?
?. How much can you assist families in helping their children do well in school?
?. How well can you implement alternative teaching strategies in your classroom?
TITLE: Verification of protein changes by parallel reaction monitoring (PRM)
AUTHORS: Lihui Li

[STEPS]
?. 200ug protein was taken from each sample of each group separately, and 10fmol heavy isotope-labeled peptide DSPSAPVNVTVR (Thermo Fisher Scientific Inc.) was incorporated as internal standard.
?. DTT was added to a final concentration of 10 mM, and then heated on the boiling water bath for 15min following cooling it to room temperature.
?. 200μL UA buffer (8M Urea, 150mM Tris-HCl, pH8.0) was then mixed in it after centrifuging at 14000 x g for 30min.
?. 100μL IAA (50mM IAA in UA) was added with 600rpm shaking for 1min.
?. 100μL NH4HCO3 buffer (50mM) was added after centrifuging at 14000 x g for 20min twice.
?. Then 40μL NH4HCO3 buffer (4μg Trypsin) was added after shaking at 600rpm for 1min.
?. 40μL NH4HCO3 buffer (50mM) was added after centrifuging at 14000 x g for 30min.
?. The filtrate was collected. The digested peptides were desalted and lyophilized, then reconstituted with 0.1% formic acid, and the peptide concentration was determined by OD280.
?. A chromatographic separation technique was performed by high performance liquid chromatography (HPLC) using Easy nLC that included Buffer A (0.1% Formic acid aqueous solution) and Buffer B (acetonitrile with 0.1% formic acid aqueous solution) (acetonitrile 84%).
?. Samples were added to the trap column and then gradient separated using a Thermo scientific EASY column at rate of 300nl/min. PRM mass spectrometric analysis was undertaken by Q-Exactive HF (Thermo Scientific Inc.).
?. The full scan was taken at a resolution of 60,000 (@m/z 200) with a scan mass range of between 300–1800m/z for 60min.
?. The AGC target was 3e6, the maximum IT was 200ms, and 20 PRM (MS2 scans) were collected according to the inclusion list after each full MS scan.
?. The secondary MS parameters included an isolation window of 1.6Th, resolution of 30,000 (@m/z 200), the AGC target was 3e6, the maximum IT was120 ms, the MS2 activation type was HCD, with normalized collision energy of 27.
?. The generated original file was analyzed using Skyline software version 3.5.0.
?. We manually checked and corrected peak selection according to MS/MS spectra, retention time, the transitions, and mass accuracy.
?. After normalizing the quantitative information by the heavy isotope-labeled peptide, a relative quantitative analysis was performed on the target peptide and the target protein.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is designed for high sensitivity miRNA library generation for the Illumina sequencing platform. Our protocol and the enhanced reagent kit enables the discovery and profiling of small RNAs from a variety of sources including FFPE, exosome, serum, and whole blood. The TailorMix workflow is designed for ease of use, enabling library preparation a single day.</p>
<p> </p>
<h3>Features:</h3>
<ul>
<li><strong>User Friendly Workflow:</strong> ­ Libraries can be prepared in a single day.</li>
<li><strong>No Additional Reagents Necessary:</strong> All reaction enzymes and buffers are provided.</li>
<li><strong>Stress Free Gel Purification:</strong> <a href="http://www.seqmatic.com/products/tailorcut-gel-extraction-tool-set/" target="_blank">TailorCut Gel Extraction Tool Set</a> is included for easy gel excision and purification.</li>
<li><strong>Easy to Use:</strong> Reagents are supplied as ready-to-use mixtures which improves consistency and reproducibility.</li>
<li><strong>Ultra High Sensitivity: </strong>Prepare miRNA libraries from any source with as little as 10ng of total RNA input<strong>.</strong></li>
</ul>

[GUIDELINES]
<p><strong>Figure 1</strong> TailorMix miRNA Sample Preparation V2 Overview</p>
<h1> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dz5effe.png" width="735" height="423" data-src="https://s3.amazonaws.com/pr-journal/dz4effe.png" data-ofn="1.png" /></h1>
<h3> </h3>
<h1><strong>Best Practices</strong></h1>
<p> </p>
<ul>
<li>Always wear gloves and use sterile technique.</li>
<li>Set up reactions using sterile non-stick nuclease-free tubes.</li>
<li>Place samples and reagents on ice or on chilled cooler block at all times and avoid extended pauses.</li>
<li>Reagents should be prepared using RNAse-free components</li>
<li>Prepare an extra 10% mixture when running multiple samples.</li>
<li>Avoid repeated freeze/thaw cycles.</li>
</ul>
<h6> </h6>
<h1><strong>RNA Input</strong></h1>
<p> </p>
<p>This protocol has been optimized using 10 - 100 ng of purified high quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. You may also use isolated miRNA as the starting material.</p>
<h1> </h1>
<h1><strong>Sample Pooling Guidelines</strong></h1>
<p> </p>
<p>The TailorMix MiRNA Sample Preparation kit is capable of multiplexing up to 96 samples into a single lane of an Illumina flow cell. While processing multiple samples in parallel, use a unique index primer for each sample at the PCR step. Samples can be pooled before or after the library purification step.</p>
<p> </p>
<h3>Citations &amp; References:</h3>
<p>Niu, Jinzhi, et al. “<a href="http://www.sciencedirect.com/science/article/pii/S0965174815300801" target="_blank">In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.</a>” <em>Insect biochemistry and molecular biology</em> 70 (2016): 127-137.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. The primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse).</p>
<p> </p>
<p>Amplicons generated using this protocol can then be sequenced using the Illumina platform.</p>
<p> </p>
<p>Primers used:</p>
<p><span style="font-weight: 400;">Fluidigm CS1+mlCOIinfF</span></p>
<p><span style="font-weight: 400;">Fluidigm CS2+HCO2198</span></p>
<p> </p>
<p><span style="font-weight: 400;">Secondary COI PCR primers</span></p>
<p><span style="font-weight: 400;">PE1-BC-CS1</span></p>
<p><span style="font-weight: 400;">PE2-BC-CS2 <br /></span></p>

[GUIDELINES]
<div>
<p>Local containment involves the employment of biological <em>safety</em> cabinets for initial specimen disinfection and <em>DNA</em> extraction. Such cabinets should not be used for <em>PCR</em> setup, as this <em>procedure</em> should be conducted apart from the aforementioned disinfection and extraction to minimize contamination.</p>
<p> </p>
<ol>
<li>Do not use any tube or plate that is not appropriate for the PCR machine you are using.</li>
<li>Make sure tubes and especially plates are well sealed before you begin run.</li>
<li>Clean up any spilled solutions and dispose of in appropriate biohazard boxes.</li>
<li>Be careful with PCR machine lids. These can be damaged if you slam or drop lids.</li>
<li>Make sure PCR heater block is clean before you start a run. Check each tube receptacle before you start.</li>
<li>Distribute tubes evenly across block so lid will seat flat against top of tubes for even heating and sealing.</li>
<li>Turn PCR machine off when you are done using.</li>
</ol>
</div>

[STEPS]
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TITLE: DNA Purification (NEB)
AUTHORS: Brian    Teague

[DESCRIPTION]
This protocol purifies DNA from an enzymatic reaction (like a PCR or a digestion with a restriction enzyme.) It is NOT an miniprep -- if you are trying to purify DNA from a culture of E. coli, you are in the wrong place!

[STEPS]
1. Make sure you are using the "Monarch PCR & DNA Cleanup Kit", not the Monarch Plasmid Miniprep Kit. They come in identical boxes -- read the label!
2. Mix the ENTIRE DNA sample with 5 times its volume of Binding Buffer. Pipette up and down several times to mix the sample thoroughly.

 

3. Insert the column into the collection tube and load the sample onto the column.
4. Centrifuge 16000 x g, 1 min. Discard the flow-through in the chemical waste container.

5. Re-insert the column into the collection tube. Add 200 µL of Wash Buffer.

6. Centrifuge 16000 x g, 1 min. Discard the flow-through in the chemical waste container.

7. Repeat steps 5 and 6 once.
8. Transfer the column to a clean 1.7 ml microcentrifuge tube.
9. Add 10 µLof Elution Buffer to the center of the silica matrix. Wait 1 min, then centrifuge 16000 x g, 1 min

10. Quantify your purified DNA on the Nanodrop.
TITLE: Polysomnography
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Polysomnography in mice

[STEPS]
1. Anesthetize the animals (ketamine and xylazine,100/10 mg/kg. i.p.) for polysomnography.
2. Screw 3 electrodes to skull above parietal and frontal cortex and cerebellum as the reference for electroencephalogram (EEG).
3. Slip 2 wire electrodes between neck muscles for electromyogram (EMG).
4. Braze thebleads to a miniature plug (Plastics One, Bilaney, Germany), cemented to skull using acrylic cement (Superbond, C&B Sun Medical) and coated using dental cement (Paladur,Heraeus Kulzer).
5. For pain caring, administer a subcutaneous injection of carprofen (5 mg/kg).
6. Once recovered, house mice in individual barrels under 12h light/dark cycle (light-on 7 a.m.) and constant temperature, tethered to the acquisition system and record 1 week out of 3 over 12 consecutive months until natural death. 
7. Based on amplified/digitized unipolar EEG and bipolar EMG signals, visually score vigilance states using a 5s sliding window frame and assign them as Waking (W), Slow Wave Sleep (SWS) and Paradoxical Sleep (PS) to produce hypnograms and state quantifications (Sleepscore, Viewpoint).
TITLE: ŌGI Bio Oxygen Flask Cleaning
AUTHORS: Jack Hocking

[DESCRIPTION]
This protocol is for the cleaning of your OGI BioReactor Oxygen Measurement Flasks.

[BEFORE_START]
Ensure protocol is carried out in sterile conditions.
Ensure you have familiarised yourself with the guidelines and warnings for this protocol.

[GUIDELINES]
If the oxygen measurement flasks are autoclaved, they should be left for 7 days before being used again and have their 100% point recalibrated prior to next use, as per our manual. They should be autoclaved at temperatures no higher than 121°C for no more than 15 minutes.
Ethanol should not be left in the flasks for more than 5 minutes.
The oxygen measurement flasks should be stored away from direct light.

[STEPS]
SECTION: Cleaning Oxygen Measurement Flask
1. Retaining the magnetic stir bar, empty the contents of each flask into the waste flask.
SECTION: Cleaning Oxygen Measurement Flask
2. Remove stir bars and wipe thoroughly with 70% ethanol.  Leave aside.
SECTION: Cleaning Oxygen Measurement Flask
5. Vigorously swill 10-20 mL of 70% ethanol around the flask.  A vortex mixer can be used if there are stubborn residues on the flask. Take care to avoid leaving the ethanol in the flask for longer than 5 minutes.
SECTION: Storage
7. Store the flasks with their stir bars inside them with the caps tightly screwed on in a dark place.  They should be left for at least 24 hours before being used again.
SECTION: Cleaning Oxygen Measurement Flask
6. Pour the ethanol from the flasks and leave to dry inverted, with the stir bars under the flasks. If using caps, spray them with ethanol and leave to dry inverted.
SECTION: Cleaning Oxygen Measurement Flask
3. Rinse out any remaining cell culture with 10-20 mL ultrapure water. A vortex mixer can be used if there are stubborn residues. N.B. ensure that the stir bar is not in the flask if vortexing, this will destroy the spot.
SECTION: Cleaning Oxygen Measurement Flask
4. Repeat Step 3 if there are still residues, dislodging with a gentle jet of water from a pipette if necessary.
TITLE: Rescue Intracranial Stenting in acute Ischemic Stroke
AUTHORS: Minh   Thang Le, Chi  Cuong Tran, Luu  Giang Nguyen, Dao  Nhat Huy Nguyen, Minh  Tuan Ngo, Hoang  Linh Duong, Minh  Luan Tran

[DESCRIPTION]
Abstract
Background
In acute ischemic stroke (AIS) caused by intracranial large vessel occlusion, rescue intracranial stenting (RIS) has been recently a treatment option to achieve recanalization in patients with the failure of mechanical thrombectomy (MT). Nevertheless, there are few studies supporting this beneficial treatment in two cerebral circulations. We aimed to analyze whether the use of RIS would improve prognosis “non-poor” of patients at 3 months.
Methods and Findings 
This was a interventional, single-arm study in patients with AIS who were treated with rescue stenting at Can Tho S.I.S hospital. Inclusion criteria consisted of: evidence of intracranial large vessel occlusion, absence of intracranial hemorrhage and severe stenosis or reocclusion after MT. Tandem lesion, loss to follow-up after discharge and a severe or fatal combined illness before AIS were excluded. The primary outcome was the “non-poor” outcome rate at 3 months and postprocedural symptomatic intracerebral hemorrhage (sICH). The study is registered with ClinicalTrials.gov, NCT04986774.Between August 2019 and May 2021, 85 eligible patients were comprised of 82 (96.5%) successful recanalization and 4 (4.7%) sICH. “Non-poor” outcome comprising of good (mRS 0 - ≤ 2) and fair (mRS 3). “Non-poor” outcome at 3 months occurred 47 (55.3%), in which there were 35 (41.2%) good outcome. DAPT was associated with new infarcts (RR = 0.1; 95%CI 0.01 - 0.7, NNT = 2) and sICH (RR = 0.1; 95%CI 0.01 - 0.9; NNT = 2). MRI 3 Tesla evaluated diagnostic occlusive lesions with sensitivity (Se) = 98.5%, positive Likelihood Ratio (LR+) = 3.5. The pc-ASPECTS < 6 points was associated with poor outcome (RR = 2.1, 95% CI 1.2- 3.7). Many predictors from demographic, history, time onset, dysphagia, imaging of MRI 3 Tesla, preprocedure, procedure and postprocedure were demonstrated the influence on poor outcome after RIS significantly (all RR > 1, all p < 0.05). The main limitations of the study was conducted in a single center, these results from clinical symptoms to imaging of MRI 3 Tesla could not only be influenced by selection bias but also not generalize to other countries in Asia.
Conclusions 
The RISIS trial suggests that RIS could be an important alternative and additional treatment afterfailureMT despite low proportion of postprocedural sICH.
Trial registration 
Clinicaltrials.gov, Identifier:NCT04986774.

[STEPS]