eamag/protocols-with-steps huggingface dataset:
TITLE: Chelex DNA extraction Protocol
AUTHORS: Sarah  Chang, Michael  Russello

[DESCRIPTION]
Laboratory protocol for DNA extraction from animal tissue with Chelex.

[STEPS]
1. Make up enough 10% Chelex for entire plate (195 µL /individual + slop) (ie. 1 g  Chelex: 9 mL  dH2O)

2. Place a magnetic stir bar in the jar containing your 10% Chelex solution. The Chelex powder is very heavy and needs to be constantly stirred in order to maintain consistency.
3. Pipette 195 µL of Chelex into each well of a 96-well plate.

4. Add approximately 1mm^2 piece of tissue (usually operculum punch or adipose fin clip) in each well. Be careful when doing this, as it is easy to mess up.

5. Add 5 µL  of proteinase K to each well using a repeater pipette. This should always be the last step, as proteinase K will digest itself at room temperature with no substrate.

6. Cover plate and incubate in the thermocycler under the following conditions: 60°C for 120 min (tissue digestion) 95°C for 15 min (heat kill for proteinase K) This protocol should be saved in most of the thermocyclers (just scroll through). Sometimes this is not sufficient for complete digestion, so a second addition of 5uL of proteinase K followed by a repeat of the incubation procedure may be necessary. Alternatively, you could increase the digestion period from 120 min to 240 min. This is what I usually do and seems to work quite well.
7. Once digestion is complete, vortex briefly to break up any remaining pieces of tissue, then vortex at 1500 rpm for 2 min to collect Chelex and any other remaining solids on the bottom of the well.
8. Pipette off the supernatant and add to a new 96 well. This will be your undiluted DNA stock that will be stored for future use. Store at -20°C in chimney well plates with caps.

9. Make up a 1:10 dilution plate to be used for the assays. Usually 200 µL  is sufficient to run all assays. Add 180 µL  of dH2O to each well, and then add 20 µL  of your undiluted stock. Vortex and spin to finish the procedure. The 1:10 dilution plate can be stored in the fridge to avoid multiple freeze/thaw cycles.
TITLE: Ligation Protocol with T4 DNA Ligase (M0202)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This product can be used for the following applications:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Cloning of restriction fragments</li><li style = "counter-reset:ol0;list-style-type:disc;">Joining linkers and adapters to blunt-ended DNA</li></ul></div></div>

[STEPS]
?. Set up the following reaction in a microcentrifuge tube .  AB1COMPONENT20 μl REACTION2T4 DNA Ligase Buffer (10X)*2 μl3Vector DNA (4 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free waterto 20 μl6T4 DNA Ligase1 μl
on ice
AB1COMPONENT20 μl REACTION2T4 DNA Ligase Buffer (10X)*2 μl3Vector DNA (4 kb)50 ng (0.020 pmol)4Insert DNA (1 kb)37.5 ng (0.060 pmol)5Nuclease-free waterto 20 μl6T4 DNA Ligase1 μl
?. Gently mix the reaction by pipetting up and down and microfuge briefly.
?. For cohesive (sticky) ends, incubate at  or  for .
16 °C
16 Room temperature
?. Heat inactivate at  for .
65 °C
?. Chill  and transform - of the reaction into .
on ice
1 µl
5 µl
[competent cells]
?. For blunt ends or single base overhangs, incubate at  or  for  (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
16 °C
16 Room temperature
TITLE: Quantitative real-time PCR for the four Lactobacillus species
AUTHORS: Chen Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Quantitative Real-time Polymerase Chain Reaction(qPCR) for the four </span><span style = "font-style:italic;">Lactobacillus</span><span> species, including </span><span style = "font-style:italic;">L.crispatus, L.gasseri, L.iners, L.jensenii.</span></div></div>

[STEPS]
?. The primers for the four Lactobacillus species used in this study were performed as Backer et al. (2007). Pick some vaginal DNA samples at random, and perform PCR reactions using these four sets of primers, respectively. Then use electrophoresis to identify the PCR products, and purify the final PCR product by gel extraction kit.
?. Use the pEASYR-T1 Cloning Kit (https://www.transgenbiotech.com/cloning_vector.html) to construct the four corresponding plasmids and identify them by sanger sequencing, respectively. After DNA quantification using Qubit Fluorometer (Life Technologies), dilute the plasmids with ddH2O by serial tenfold dilution into 6 concentration gradients as serial standards. The copies of standards (copies/μl)= (DNA concentration/ (number of base pairs*324))*6*1014. The number of plasmid base pairs (bp) of L.iners, L jensenii, L. crispatusand L. gasseri are 4082, 4013, 4082, 4251.
?. Use SYBR Premix Ex Taq GC (TAKARA) to conduct the qPCR. The reaction mixture contains 10 μl of 2×SYBR Premix Ex Taq GC, 0.2 μM forward primer, 0.2 μM reverse primer, 1.6 μl of DNA sample and 8.2 μl ddH2O to make up the final reaction volume of 20 μl. Each reaction is run in triplicate.
?. Use the StepOnePlus Real-time PCR System (Life Technologies) to conduct the qPCR. The condition of qPCR amplification is followed as Table above. The standard curve range of amplification efficiency for the qPCR is from 90 to 110%, and linearity values is ≥0.99. In case the result was not in the range of the standard curve, the samples are diluted tenfold and analyzed in triplicate again. The average log10 copies/ml are expressed as per 1 ml urine sample.
?. Then transfer all the final reaction volumes into a 96-well plate. The DNA templates in each plate contain urine samples, corresponding serial standards and negative controls (ddH2O).
TITLE: TEST - manual DNA Purification via magnetic beads 
AUTHORS: Carla Jungkunz

[DESCRIPTION]
manual purification of DNA/PCRs via magnetic beads 

[BEFORE_START]
bring magnetic beads to room temperature (at least 30min)

[STEPS]
SECTION: prepare magnetic beads
1. 30 min bring magnetic beads to room temperature (RT)
SECTION: prepare magnetic beads
2. vortex until homogeneous 
SECTION: Binding of DNA
3. Add beads                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            0.9 times the amount 
SECTION: Binding of DNA
4. 5 min incubate for at least 5min
SECTION: Binding of DNA
5. 5 min place tube in magnetic rack 
SECTION: Binding of DNA
6. remove supernatant
SECTION: 1st wash
7. add ethanol 
1.9 times the amount 
vortex thoroughly
SECTION: 1st wash
8. 5 min place in magnetic rack 
SECTION: 1st wash
9. remove supernatant
SECTION: 2nd wash
10. add ethanol 
1.9 times the amount 
vortex thoroughly
SECTION: 2nd wash
11. 5 min place in magnetic rack 
SECTION: 2nd wash
12. remove supernatant
SECTION: Airdry beads
13. 10 min keep lid open in magnetic rack
SECTION: Airdry beads
14. remove residual supernatant
SECTION: Elute DNA
15. place tube in sample rack 
SECTION: Elute DNA
16. add indicated volume of water and mix/vortex thoroughly
5 min incubate 
SECTION: Elute DNA
17. 5 min  place in magnetic rack 
SECTION: Elute DNA
18. transfer supernatant into labeled tube
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>Use with Ultra Streptavidin Detection Kit (<a href="https://antibody.biolegend.com/datasheet.php?UpProd=&amp;catalogno=SIG-32250" target="_blank">SIG-32250)</a> or (<a href="https://antibody.biolegend.com/datasheet.php?UpProd=&amp;catalogno=SIG-32248" target="_blank">SIG-32248)</a></p>
<p> </p>
<p>Positive control: Normal human cerebellum (except SMI-71, which should be rat brain)</p>

[STEPS]
SECTION: Clear Slides
?. 
SECTION: Rinse slides
?. 
SECTION: Antigen Retrieval
?. 
SECTION: Antigen Retrieval
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SECTION: Antigen Retrieval
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SECTION: Antigen Retrieval
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SECTION: Counterstain
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SECTION: Counterstain
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SECTION: Counterstain
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SECTION: Counterstain
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SECTION: Clear slides
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SECTION: Coverslip
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TITLE: Making Agarose for use in acute in vivo Electrophysiology Experiments 
AUTHORS: Ryan Gillis, Mikayla Carlson, Severine Durand

[DESCRIPTION]
This protocol describes the procedure for making agarose used in acute in vivo Electrophysiology Experiments with Neuropixels probes. This exact methodology is crucial to the the success of these types of experiments due to the requirements that the agarose must be strong enough to provide the brain stability while also being penetrable by the delicate probes. This agarose preparation protocol is used in Neuropixels Data Collection: Whole Hemisphere Recordings and Visual Cortex Recording Window Replacement for in vivo Elctrophysiology Experiments.

[STEPS]
SECTION: Create Agarose Aliquot
1. Place a piece of weigh paper on scale  
SECTION: Create Agarose Aliquot
2. Make sure the scale is tared and display is showing grams.
SECTION: Create Agarose Aliquot
3. Measure out .42 g of Certified Low-Melt Agarose and transfer powder to 2mL centrifuge tube.

SECTION: Create Agarose Aliquot
4. On the same paper, measure .40 g of High-EEO Agarose.

SECTION: Create Agarose Aliquot
5. Carefully transfer agarose powder to the same 2mL centrifuge tube and seal tightly. 
 

SECTION: Prepare Agarose
8. Add the contents of one aliquot to 125-mL an autoclaved Erlenmeyer flask along with 20.2 mL ACSF.V.

 

SECTION: Prepare Agarose
9. Mix Agarose and ACSF.V with stainless metal stir stick, taking care not to get the mixture on edges of flask.
SECTION: Prepare Agarose
10. Heat in microwave for 67 s on power 3 (30% of full power) then stir well to avoid bubbles in the mixture.

SECTION: Prepare Agarose
11. Heat for 15 s on power 2 (20% of full power) then stir well.

SECTION: Prepare Agarose
12. Heat for 15 s  on power 1 (10% of full power) then stir well. Ensure mixture is clear with no visible bubbles or granularity.



SECTION: Prepare Agarose
13. Pour agarose mixture slowly and carefully into 10ml autoclaved graduated cylinder to avoid agitation.
 


 


SECTION: Prepare Agarose
14. Store the 10ml cylinder in a water bath at 50 °C . Ensure water level in the bath is approximately even with the height of the agarose in the cylinder. Agarose can be stored in a hot water bath for at least 12 hours. After that, check for signs of drying, abnormal coloration, or excess bubbles before using.
 



SECTION: Create Agarose Aliquot
6. Repeat Steps 1-5 to make as many aliquots as desired.
SECTION: Create Agarose Aliquot
7. Store aliquots in sealed centrifuge tubes at room temperature. They can be stored up to a year, provided they stay dry and are not exposed to any extreme conditions.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Reaction Conditions and Buffer, Materials:</strong><br /><br />
<ul>
<li>Reaction conditions: 50 mM Tris-HCl, pH 7.8, 10 mM EDTA, 0.5 mM DTT, 2 µCi Adenosyl-L-Methionine, S-Methyl (<sup>3</sup>H), 3 µg DNA</li>
</ul>
<br />
<ul>
<li>Preparation of reaction buffer, 10X</li>
</ul>
<p>               3000.0 µL of 1 M Tris-HCl, pH 7.8</p>
<p>               1200.0 µL of 500 mM EDTA, pH 8.0</p>
<p>               1500.0 µL of d-H<sub>2</sub>O</p>
<p>               To 95.0 µL of the reaction buffer, add 5.0 µL of 100 mM DTT.</p>
<p>               Use 5.0 µL per 50.0 µL reaction.<br /><br /></p>
<ul>
<li>Proteinase K, 2 mg/mL (autodigested at 37°C for 60 min)</li>
</ul>
<p> </p>
<ul>
<li>ss salmon sperm DNA, 5 mg/mL</li>
</ul>
<p> </p>
<ul>
<li>20% TCA</li>
</ul>
<p> </p>
<ul>
<li>5% TCA</li>
</ul>
<p> </p>
<ul>
<li>500 mM NaOH</li>
</ul>

[STEPS]
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TITLE: Akoya Biosciences PhenoCycler Fusion (formerly CODEX) User Guide
AUTHORS: Andrew Houston

[DESCRIPTION]
Link to PhenoCycler User Guide to allow for multi-plex spatial proteomic detection on fresh frozen and FFPE tissue sections

[STEPS]
1. Staining/slide prep protocol can be found here:
  

FFPE tissue staining pgs. 45-56
Reporter plate preparation pgs. 70-75

2. Imaging protocol can be found here:
  

Flow cell assembly pgs. 29-39
Instrument set-up for PhenoCycler scan pgs. 57-71
TITLE: Total nucleic acid extraction - Maxwell(R) HT Environmental TNA Kit, custom (Promega)
AUTHORS: Adélaïde Roguet

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Total nucleic acid extraction from wastewater using Maxwell(R) HT Environmental TNA Kit, custom (Promega)</div></div>

[STEPS]
?. [Total nucleic acid extraction]
For HA filter extraction, let the sample thaw on ice and go to step 2. For BCoV/BRSV extraction (in duplicate), add 5 µL of BCoV/BRSV solution to the 2-mL tube containing 250 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.For Direct extraction, add 150 µL of wastewater to the 2-mL tube containing 100 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.
?. [Total nucleic acid extraction]
For HA filter extraction, place the 2-mL tubes in the bead beater.
?. [Total nucleic acid extraction]
Bead beat for
Start the bead beating when the beads start to be loose in the tubes.
?. [Total nucleic acid extraction]
Cooldown the samples on ice for  .
?. [Total nucleic acid extraction]
Repeat Steps 9.1 and 9.2 once .
?. [Total nucleic acid extraction]
Centrifuge at maximum speed for 1 min at room temperature.
Centrifuge: 150000 33, 1 min
?. [Total nucleic acid extraction]
For HA filter extraction, transfer 125-250 µL of supernatant to the Lysis/Bind plate. For BCoV/BRSV/Direct extraction, transfer all supernatant to the Lysis/Bind plate.
The default volume transferred is 250 µL. However, for WWTPs with "dirty" influents, we only transfer 125 µL.
?. [Total nucleic acid extraction]
Start the protocol Promega_Maxwell_HT_RNA_Wastewater_V1.bdz on the KingFisher Flex
?. [Total nucleic acid extraction]
Transfer the purified sample from the Elution plate to the microcentrifuge tubes.
The DNA/RNA is now ready for downstream applications. RNA extract may be stored in RNase-free water at -80ºC for 1 year.
?. [RT-ddPCR]
Quantification by Droplet Digital PCR (ddPCR)dx.doi.org/10.17504/protocols.io.bpg6mjze
TITLE: Preparing multiplexed WGS/MetaG  libraries with the Illumina DNA Prep  kit for the Illumina NextSeq or MiSeq
AUTHORS: André M Comeau, Gina V Filloramo

[DESCRIPTION]
The preparation of (meta)genomic libraries using the Illumina Nextera Flex (now "DNA Prep") kit with CD/UDI indices at the IMR.

Based upon Illumina protocol 10000000254 16 v10 (Aug.2021).

[STEPS]
SECTION: Prepare and Quantify gDNA
1. Using two 96-well PCR plates, prepare 30 µL of 1:10 dilution using nuclease-free water (NFW) for up to 192 gDNA samples. 

SECTION: Prepare and Quantify gDNA
2. Quantify 5 µL of the 1:10 diluted gDNA using the Invitrogen Quant-iT dsDNA HS assay kit on the fluorescent 96-well plate reader.

 

SECTION: Perform Illumina DNA Prep Kit Library Preparation
3. Follow the library preparation instructions as described for the Illumina DNA Prep kit (https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/illumina_prep/illumina-dna-prep-reference-guide-1000000025416-10.pdf) with the below modifications.

 

SECTION: Perform Illumina DNA Prep Kit Library Preparation
3.1. Amplify Tagmented DNA: use 12 PCR cycles (based on 5 ng of input DNA).
SECTION: Perform Illumina DNA Prep Kit Library Preparation
3.2. Final library clean-up: wash beads with 80% ethanol as described, remove excess ethanol and allow beads to dry on the magnet for 5 min. Remove the plates from the magnet and add  60 µL of Resuspension Buffer (RSB) to the beads. Pipette mix until thoroughly resuspended and incubate at room temperature for 2 min. Place the plates back on the magnet until clear and transfer 55 µL of the supernatant into new 96-well plates.


SECTION: Quantify and Pool Final Library
4. Quantify 5 µL of each tagmented, amplified and purified sample using the Invitrogen Quant-iT dsDNA HS assay kit on the fluorescent 96-well plate reader. 
SECTION: Quantify and Pool Final Library
5. Pool equal molar amounts of up to 192 samples in a 1.5 mL microcentrifuge tube. 
SECTION: Quantify and Pool Final Library
6. Dilute pooled library with RSB to the recommended loading concentration specific to the intended sequencer per Illumina's guidelines.
TITLE: UC Davis - Immunohistochemistry BODIPY
AUTHORS: Jennifer Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids. It has uses for identifying and quantifying lipid droplets in various cell and tissue type. Abnormal lipid droplet accumulation can be associated with various metabolic disorders such as Diabetes, cardiovascular disease and cognitive impairment. </div></div>

[STEPS]
?. Frozen tissue sections (10µM) on slides (tissues embedded in OCT) are then fixed in 4% paraformaldehyde for 5 minutes. a. Alternatively paraffin embedded sections are deparaffinized through 3 100% xylene 3 min wash b. Sections were rehydrated through 100%, 95%, 70%, 50%, and 30% then into deionized water (3min each). c. Antigens are unmasked by steaming in Sodium citrate for 10 min.
?. Wash three times for ten minute in PBS
?. The sections where then incubated in 0.1mg/ml BODIPY® 492/515 in DMSO for 30 minutes in the dark.
?. Wash three times for ten minute in PBS
?. Coverslip mounted with Prolong gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI).
TITLE: Hematoxylin & Eosin Protocol For Leica ST5020 Automated Stainer
AUTHORS: Angela Denn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This Hematoxylin and Eosin protocol is for the Leica ST5020 Automated multistainer. You can use it as a guide to developing your own protocol, catered to the needs of your researchers. This template can be modified for manual staining but best and most reproducible results are easier to achieve with an automated stainer. </div></div>

[STEPS]
?. [H&E Stain]
ABCDEF1STEPSTATIONDYE/REAGENT STATIONDURATIONEXACTDIP2138Oven station5:00nono3237Oven station5:00nono431Pro-Par5:00noyes542Pro-Par5:00noyes653Pro-Par5:00noyes764100% IPA1:30noyes875100% IPA1:30noyes98690% IPA1:30noyes10912DI Water Wash1:30nono111021SelectTech Hematoxylin 5601:30yesyes121111DI Water Wash1:30nono131233SelectTech Define1:00yesno141310DI Water Wash2:00nono151432SelectTech Blue Buffer 81:30yesno16159DI Water Wash1:30nono17163170% IPA1:00noyes181718ST Alcoholic Eosin Y 5150:10yesyes19182990% IPA0:20yesyes201917100% IPA0:30yesyes212016100% IPA1:00noyes222115100% IPA1:30noyes232228*100% IPA1:30noyes242327*100% IPA1:30noyes252426*50:50 Pro-par/100% IPA1:00yesyes262514*Pro-Par5:00noyes272613Pro-Par5:00noyes2827EXITPro-Par
ABCDEF1STEPSTATIONDYE/REAGENT STATIONDURATIONEXACTDIP2138Oven station5:00nono3237Oven station5:00nono431Pro-Par5:00noyes542Pro-Par5:00noyes653Pro-Par5:00noyes764100% IPA1:30noyes875100% IPA1:30noyes98690% IPA1:30noyes10912DI Water Wash1:30nono111021SelectTech Hematoxylin 5601:30yesyes121111DI Water Wash1:30nono131233SelectTech Define1:00yesno141310DI Water Wash2:00nono151432SelectTech Blue Buffer 81:30yesno16159DI Water Wash1:30nono17163170% IPA1:00noyes181718ST Alcoholic Eosin Y 5150:10yesyes19182990% IPA0:20yesyes201917100% IPA0:30yesyes212016100% IPA1:00noyes222115100% IPA1:30noyes232228*100% IPA1:30noyes242327*100% IPA1:30noyes252426*50:50 Pro-par/100% IPA1:00yesyes262514*Pro-Par5:00noyes272613Pro-Par5:00noyes2827EXITPro-Par
?. [H&E Stain]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol details the steps to annotate Anvi'o gene calls for function using Uproc. </p>

[STEPS]
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TITLE: Axenic Diatoms cultures protocol
AUTHORS: Mariella Ferrante, Rossella Annunziata, Monia Teresa Russo, Francesco Manfellotto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Axenic cultures protocol</div><div class = "text-block">Axenicity of cultures is obtained by multi-antibiotic treatment:  by adding antibiotics directly to the medium in which the strain will be inoculated. After a variable amount of days we refresh the coltures with new medium.</div></div>

[STEPS]
?. [antibiotics concentration]
The antibiotics used are ampicillin, streptomycin and penicillin; We used the following final concentration:Ampicillin 0,7 mg/mlStreptomycin 0,1 mg/mlPenicillin 0,5 mg/ml
?. [antibiotics concentration]
Add antibiotics in F/2 medium.Start from 20/50.000 cells in 25 ml of F/2 medium with antibiotics.Refresh 3 times the cultures every 3 days with new medium with antibiotics.
?. [antibiotics concentration]
Transfer the cultures into a variable higher volume flask containing F/2 medium and antibiotics.Arrive at the desired final volume.
?. [contamination test ]
Assess axenicity of the strains by fluorescence microscopy using DAPI staining (4',6-diamidino-2-phenylindole-a DNA stain).(DAPI stains DNA, if bacteria are present in the culture bacterial nucleoids can be visualised as fluorescent spots).To fix cells use neutralized formaldehyde 1.6% DAPI final concentration 1:1000
TITLE: U Mass - Glucose Tolerance Test
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Glucose tolerance test measures systemic clearance of glucose following an intraperitoneal bolus injection of 20% dextrose. This experiment measures insulin sensitivity in awake mice assuming that there are no alterations in the animal’s pancreatic -cell function and insulin secretion. Insulin sensitivity is altered in obese mice.</div></div>

[STEPS]
?. Mice may be fasted overnight (~15 hours) or for 5 hours prior to the start of experiment.
?. Collect plasma sample (10 µl) before the start of experiment (basal-0 min) to measure basal glucose levels.
?. Administer intraperitoneal injection of 20% dextrose (1 or 2 g/kg body weight) using an insulin syringe.
?. Collect plasma samples (10 µl) at 10, 20, 30, 60, 90, and 120 min following injection to measure circulating glucose concentrations.
?. For data analysis, plasma glucose levels vs. time after injection are plotted, and areaunder-curve may be calculated to estimate insulin sensitivity.
?. Area-under-curve of glucose tolerance test may be inversely correlated with insulin sensitivity assuming unaffected insulin secretion and pancreatic β-cell function in mice.