eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Ligation protocol for the&nbsp;PCR Cloning Kit (E1202)

[BEFORE_START]
For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. See the Guidelines for the formulas.

[GUIDELINES]
<br /><table border='0' width='100%' cellspacing='0' cellpadding='0'><tbody><tr><td>&nbsp;</td><td align='right'><strong>LIGATION REACTION</strong></td><td align='right'><strong>POSITIVE CONTROL</strong></td></tr><tr><td>Linearized pMiniT Vector (25 ng/&mu;l)</td><td align='right'>1 &mu;l (25 ng)</td><td align='right'>1 &mu;l (25 ng)</td></tr><tr><td>Insert*</td><td align='right'>1&ndash;4 &mu;l*</td><td align='right'>&ndash;</td></tr><tr><td>Amplicon Cloning Control (1 kb) (15 ng/&mu;l)</td><td align='right'>&ndash;</td><td align='right'>2 &mu;l (30 ng)</td></tr><tr><td>H2O</td><td align='right'>to 5 &mu;l</td><td align='right'>2 &mu;l</td></tr><tr><td>Cloning Master Mix (2X)</td><td align='right'>5 &mu;l</td><td align='right'>5 &mu;l</td></tr><tr><td>Total Volume</td><td align='right'>10 &mu;l</td><td align='right'>10 &mu;l</td></tr></tbody></table><br class='Apple-interchange-newline' />*For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. Formulas below use the recommended values of 25 ng of linearized vector (2525 bp) per reaction and an insert-to-vector ratio of 3:1.<br /><br /><strong>a.</strong> Relative length calculations:<br />ng insert to be added = (3)(25 ng vector) (bp of insert/2525 bp of vector)<br /><br /><strong>b.</strong> Molar calculations:<br />&nbsp; &nbsp; &nbsp;i. &nbsp; Convert the 25 ng vector present in the ligation reaction to pmoles:<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; (25 ng vector)(1000)/(650 daltons per base pair)(number of base pairs in vector or<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 2525) = (25)(1000)/(650)(2525) = 25000/1641250 = 0.015 pmoles vector<br />&nbsp; &nbsp; &nbsp;ii. &nbsp;Calculate a 3-fold molar amount of insert to add to each ligation:<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; (3)(0.015 pmoles vector) = 0.045 pmoles insert<br />&nbsp; &nbsp; &nbsp;iii. Convert the pmoles insert amount to ng insert to be added:<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;ng insert to be added = (0.045 pmoles insert)(base pairs in insert)(650 daltons per<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;base pair)/1000<br /><br />As examples, these calculations will yield insert levels of 15 ng (500 bp insert), 30 ng (1 kb insert) or 60 ng (2 kb insert).<br /><br />For unpurified PCR amplicons from reactions yielding a specific product, use 1 &mu;l or less of the reaction as insert. Do not use larger amounts, as carryover material from the PCR reactions can inhibit ligation or transformation. To estimate the concentration of PCR product for the above calculations, compare the reaction yield to known amounts of DNA fragments in a marker lane, such as our Quick-Load&reg; Purple 2-Log DNA Ladder (<a href='https://www.neb.com/products/n0550-quick-load-purple-2-log-dna-ladder-0-1-10-0-kb'>NEB #N0550S</a>).

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is widely used in serum biochemical indexes detection, it provides an rapid and reliable technique for obtaining relative concentrations of multiple blood biochemical indices simultaneously.</p>

[BEFORE_START]
<p>All the standard samples of every index are kept at  4 ℃.</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for DNA Assembly using the&nbsp;NEBuilder&reg; HiFi DNA Assembly Master Mix (E2621).

[GUIDELINES]
<strong>Optimal Quantities</strong><br /><br />NEB recommends a total of 0.03&ndash;0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2&ndash;0.5 pmols of DNA fragments when 4&ndash;6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool,<a class='ga-event' href='https://www.neb.com/external-links/nebiocalculator' target='_blank' data-ga-category='External Link' data-ga-action='https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol' data-ga-label='https://www.neb.com/external-links/nebiocalculator'>NEBiocalculator</a>.<br /><br />pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)<br />50 ng of 5000 bp dsDNA is about 0.015 pmols<br />50 ng of 500 bp dsDNA is about 0.15 pmols<br /><br />The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.

[STEPS]
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TITLE: Planetary Dominoes - How microbes drive biogeochemical cycles
AUTHORS: Nadia Szeinbaum, Abbigail Johnson, Thomas Swofford

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the step to assemble your own Planetary Domino. </div><div class = "text-block"><span>We designed this at Georgia Tech for an Astrobiology outreach event called "</span><a href="https://www.facebook.com/events/1731095390302446/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">An Evening of Wonder</span></a><span>", which we organized as part of </span><a href="http://abgradcon.org/old_sites/abgradcon2018/index.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">AbGradCon2018</span></a><span>. The goal of this activity was to introduce how microorganisms drive many geochemical cycles and how they are interconnected. Each domino piece corresponds to a bacerial functional role/"trophic" role (i.e., aerobic heterotrophs, anaerobic metal reducers, etc.) contains substrates on one side and products on the other. The idea is to find how they can be connected, like pieces of a domino.   </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div>

[STEPS]
?. Print this file into a poster, cut each piece and add an adhesive magnet. These will be the domino pieces, and they are now able to stick to any magnetic surface. The pieces here are enough for at least three different sets, as they repeat. The design was created by Image Alleviation, in collaboration with us, specifically for this project.
?. After a brief introduction about microbes in the environment (good vs. bad, or biogeochemical cycles, depending on the audience), ask your audience to get into groups or one by one add pieces of the domino to the board. It is key is to let them discuss and ask questions, and only if necessary explain why certain pieces might not go together, etc.
TITLE: Conseils sur l&#39;utilisation de contrôles d&#39;analyse pour la détection directe des poliovirus par séquençage des nanopores
AUTHORS: Joyce Akello, Manasi Majumdar, Alex Shaw, Catherine Troman, Erika Bujaki, Javier Martin, Nick Grassly

[DESCRIPTION]
Ce document décrit le processus de laboratoire pour l'utilisation de contrôles d'analyse (contrôles positifs et négatifs) pour effectuer la détection directe du poliovirus par séquençage des nanopores (le test DDNS).

[STEPS]
TITLE: Intracardiac perfusion and brain fixation for immunohistochemistry
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
This protocol aims to preserve brain tissue for immunohistochemistry studies. It is not valid for protein or RNA extraction studies.

[BEFORE_START]
Prepare o filtered fresh PFA and saline solution (or PBS).

[GUIDELINES]
Read the whole protocol before starting the procedure.  The whole process last about 3 days.


[STEPS]
SECTION: Animal Sacrifice
1. Before starting, fill a syringe with 20 mL of ice-cooled PBS and a separate syringe with  20 mL 4% Paraformaldehyde (PFA). Connect the PBS syringe to a Winged infusion set.
SECTION: Animal Sacrifice
4. 
When perfusion is finished, harvest the brain from the cranium, carefully removing the meninges to avoid damage to the tissue. 


SECTION: Brain post-fixation and cryoprotection
5. Submerge the brain in a 15 mL falcon tube (or similar) containing 12 mL of 4% PFA for 960 min at 4 °C .
 

SECTION: Brain post-fixation and cryoprotection
6. Wash the brain 3 x 5 min  in the Falcon tube, with agitation, employing 4 °C PBS or TBS to remove PFA traces completely.

SECTION: Brain post-fixation and cryoprotection
7. Thereafter,  place 12 mL of 15 Mass / % volume sucrose in the container and place it at 4 °C until the tissue sinks. At this point, the experimenter should see that the brain floats on the liquid surface.
 

SECTION: Brain post-fixation and cryoprotection
8. When the brain has sunk, discard the sucrose and add 12 mL of  30 Mass / % volume sucrose. Place the tube again at 4 °C until the tissue sinks. 
 

SECTION: Animal Sacrifice
2. Anesthetize the animal deeply. Ensure there are no reflexes and breathing is slow. Carefully cut the thoracic cavity until the heart is exposed.
SECTION: Animal Sacrifice
3. To perform intracardial perfusion, puncture the ventral region of the right ventricle with the winged infusion set and sustain the needle firmly. Carefully cut (make a small opening) the left atrium to facilitate fluid outflow. Perfuse the animal with 20 mL of cold on ice PBS. Change the syringe in the winged infusion set, and continue the perfusion with  20 mL of cold on ice 4% PFA.
 

SECTION: Brain freezing
9. Prepare and label plastic or crystal containers to store the brain for the long-term at -80 °C . The containers and forceps to hold the brains must be cooled in dry ice.  
SECTION: Brain freezing
10. Discard the sucrose and extract the brain from the Falcon tube. Roll the brain on clean absorbent tissue paper to clean sucrose traces. Let the brain air-dry for 2 min.
SECTION: Brain freezing
11. To freeze the brain, place it on top of aluminum foil in a container filled with dry ice for 4 min . Alternatively, the brain can be wrapped in aluminum foil and submerged in liquid nitrogen for 8 s 
 

SECTION: Brain freezing
12. Using the dry-ice-cooled forceps, place the brain into the dry-ice-cooled container and store it at -80 °C for further processing.
TITLE: DNA extraction - Zooplankton - 96 wells
AUTHORS: coline.royaux, Nicolas Rabet, Céline Bonillo

[DESCRIPTION]
This protocol was used to extract DNA from whole or parts of zooplanktonic freshwater crustaceans (Copepoda, Branchiopoda, ...) from New Caledonia.

[STEPS]
1. Prepare your 96-well extraction plate with one individual per well. Alternate genus in the wells to detect eventual contamination between wells.
1.1. Collect one individual from a sample
1.2. Note its genus and determine its sex with a binocular microscope
1.3. For big individuals (more than 5 mm), dissect a few legs and put it in the well. Be careful not to damage the rest of the body and put it in a tagged Eppendorf tube.
For little individuals (less than 5 mm), put the whole body.
 

1.4. When all 96 wells are filled, the biological material has to dry to go to lysis

 

 



2. Prepare the lysis
2.1. Mix 18 mL and 2.5 mL in a Multi-Channel Reservoir and distribute 200 µL with a multimicropipette in each well
 

2.2. Close your extraction plate with a heated aluminium foil and an adhesive plastic film
3. Put your plate in a proofer at 56 °C    to lyse the tissues
4. Perform the DNA extraction with a DNA extraction robot
4.1. Remove the adhesive film and aluminium foil from the plate and put it in the robot
4.2. It will deposit 200 µL and 200 µL in each wells and mix it

4.3. Then, 600 µL are transfered on the tissue binding plate. Reagents excess are emptied in a waste container.

4.4. The tissue binding plate is then dried by a 5 min aspiration to bind DNA to the silica membrane of the binding plate
4.5. The silica membrane is then washed with 600 µL and twice with 900 µL per well. Each wash is intercalated by a 5 min aspiration dry.

4.6. The waste container is then removed from under the binding plate which is dried again by a 10 min aspriration

4.7. An empty extraction plate is placed under the binding plate to retrieve the genomic DNA from it
4.8. DNA is eluted from the tissue binding plate with 100 µL in each well and is collected in the new extraction plate underneath

4.9. After a 3 min rest, the binding plate is dried for 2 min and the elution is repeated with 100 µL 

4.10. Retrieve the new extraction plate containing the genomic DNA and discard the rest
TITLE: Preparation of PBS Solution
AUTHORS: Stephane  Fadanka, Shalo  Minette, Nadine    Mowoh

[DESCRIPTION]
Phosphate buffered saline ( PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. This SOP gives one of the most common formulations used in preparing PBS (1x PBS).

137 mM NaCl
10 mM Na2HPO4
1.8 mM KH2PO4 
pH 7.2 -7.4

PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface. It is applied in washing cells after protein expression procedures due to its osmolarity and pH to avoid cell disruption to preserve and subsequently freeze the cells for downstream molecular biology applications. 




[BEFORE_START]
Make sure all salts and equipment to be used are available in the right quantities and functional respectively.

[STEPS]
SECTION: Preparing reagents and workspace
1. Before beginning the procedure put on Personal Protective Equipment (Lab Gown, Gloves, shoes, masks and goggles).

Clean up work surfaces and environment using disinfection solution (Bleach and after 70% alcohol).

Check to be sure all reagents and equipment needed for the procedure are available, in place and functional.

SECTION: Weighing and dissolving salts
2. Use an electronic balance to accurately weigh out all salt powders  ,   and    into an appropriate size weighing boat with   into an appropriate size weighing boat with the help of a spatula in recommended amounts as indicated in the table below.

Composition of 1X PBS buffer


 
 
    SaltConcentration (mmol/L)Concentration (g/L)Amount for 500 mlNaCl1378.04.0Na2HPO4101.420.71KH2PO41.80.240.12
Transfer the contents of the weighing boat into a 500ml or larger beaker.
 
 Use a 100ml measuring cylinder to measure 100ml of sterile distilled water and pour carefully to rinse the boat so as to take out all the powder into the beaker.

Mix gently by swirling and take the beaker onto a magnetic stirrer  and put the magnetic flea for about 3 minto completely dissolve the salts.
SECTION: Weighing and dissolving salts
3. Use a pH meter to measure the pH of the solution and either add  or   to adjust the to pH 7.2or pH 7.4as desired.
Measure out the appropriate amount of sterile distilled water to make up the volume of the solution to 500ml of 1x PBS.
SECTION: Sterilisation and storage
4. Transfer the contents of the beaker into a 500ml or 1000ml Duran Bottle and autoclave to make the PBS solution sterile. 
SECTION: Sterilisation and storage
5. Remove the 1x PBS from the autoclave, allow it to cool, cork tightly and store at 23 Room temperature or in the refrigerator and store at 4 °C. The 1x PBS buffer is ready for use in any molecular biology technique.
TITLE: Primer stock preparation
AUTHORS: Brian Lovett, Kristen Pierce

[DESCRIPTION]
General protocol for preparation of lyophilized primer stocks from IDT.

[GUIDELINES]
Recommended: When you receive your lyophilized primers, check the name of the primer, primer sequence and the concentration listed on the tube. IDT provides a specification sheet you can download/archive for more information.

[STEPS]
SECTION: Preparation of stock solution
1. Reconstitute lyophilized primers in required volume of molecular grade water (i.e., RNase/DNase free) to 100 micromolar (µM).

SECTION: Preparation of working solution
4. Allow stock solution to thaw completely at Room temperature.
SECTION: Use of primers
8. For a typical 25 µL PCR reaction, 1 µL of each primer is used (i.e., 0.4 micromolar (µM) final concentration).
SECTION: Preparation of stock solution
2. After water has been added, vortex for at least 10 s to ensure the lyophilized primers are fully dissolved.
SECTION: Preparation of stock solution
1.1. Practically, the microliters of water required to create a 100 micromolar (µM) solution is 10x the nanomoles of lyophilized primer (i.e., if 23.5 nanomoles is noted on side of tube, then add 235 µL of water to stock tube).

SECTION: Preparation of stock solution
3. Once reconstituted, the stock solution can be stored in a labeled box at -20 °C, and thawed completely before use.

SECTION: Preparation of working solution
5. Vortex the stock solution for at least 10 s to ensure homogenization before adding it to the working solution. 
SECTION: Preparation of working solution
7. Once the concentrated stock is added, vortex the new working solution for at least 10 s and store at -20 °C to use as needed.  
SECTION: Use of primers
8.1. To maintain optimal use of your primers, limit freeze/thaw cycles, as this can lead to degradation over time. 
SECTION: Preparation of working solution
4.1. While waiting for your stock to thaw, prepare clean microcentrifuge tubes by labeling them with the primer name and date of preparation. Label the lid so it is easy to identify tubes in freezer box.
SECTION: Preparation of working solution
6. To prepare a 10 Molarity (m) working solution from the 100 micromolar (µM), you will dilute 1:10  (i.e., 1 µL of stock for every 9 µL of water). 

SECTION: Preparation of working solution
6.1. For a 100 µL working solution, pipette 90 µL of molecular grade water into the newly labeled microcentrifuge tube and then add 10 µL of the stock solution.
TITLE: Find SARS-CoV-2 testing protocol
AUTHORS: jose.nuno 

[STEPS]
?. Collect a sample of   of saliva (up to the filling line) in a 0.45 um syringeless filter vial (provided with the kit). The sample should be collected at least 60 minutes after the last food ingestion and after a mouth rinse with plain water to clean the mouth
400 µl
Be careful of cleaning the surface of the vial if saliva overflows the vial or if saliva particles are expelled from the tube. All saliva samples at this stage must be handled as biohazard materials
All saliva samples should be processed in the following 60 minutes after being generated
?. Put the filter head in the vial and push the plunge until reaching a full stop
Be careful of cleaning the surface of the vial if saliva overflows the vial or if saliva particles are expelled from the tube. All saliva samples at this stage must be handled as biohazard materials
All saliva samples should be processed in the following 60 minutes after being generated
?. Open the lid on the vial plunge and take a  saliva sample with the fixed volume capillary tube provided with the kit
15 µl
Be careful of cleaning the surface of the vial if saliva overflows the vial or if saliva particles are expelled from the tube. All saliva samples at this stage must be handled as biohazard materials
All saliva samples should be processed in the following 60 minutes after being generated
?. Put the  filtered saliva sample in the testing site of the testing card
15 µl
Be careful of cleaning the surface of the card if saliva overflows the testing site or if saliva particles contaminate the surface of the card. All saliva samples at this stage must be handled as biohazard materials
All saliva samples should be processed in the following 60 minutes after being generated
?. Wait  seconds
?. Peel out the adhesive tape covering the exhaust perforations in the card
?. Wait  minutes for the reaction to fully occur
?. Open the Xplora app, follow the steps to take the photograph and read the final result from the smartphone screen
TITLE: Removal of kanamycinR gene from Keio collection strain
AUTHORS: Ben Kuipers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to remove the kanamycinR gene from the JW3367 strain from the Keio Collection.</div><div class = "text-block">References Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97: 6640-6645. </div><div class = "text-block">Barrick Lab. (16 May, 2014). FLP Recombination in E. coli. Retrieved 3 September, 2016, from http://barricklab.org/twiki/bin/view/Lab/ProcedureFLPFRTRecombination</div><div class = "text-block">T--Hong_Kong_HKUST--Protocol_RemoveKAN.pdf</div></div>

[STEPS]
?. Chemical competent cells of E. coli JW0336 were prepared with the mix and go protocol.
?. Day 1: Transformation of the recombinase plasmid pCP20The competent cells were transformed with plasmid pCP20. (This plasmid has a temperature-sensitive origin of replication, resistant to ampicillin and chloramphenicol. With the presence of the FLP recombinase, the ampicillin and chloramphenicol resistance genes would be removed under the temperature higher than 43ºC).1 ml LB was added for recovery which lasted for 1 hour.The eppendorf was centrifuged for 2.5 minutes with 11000xg. Supernatant was discarded and the pellet was resuspended and plated on the LB + AMP plate.The plate was then incubated for 1.5 days under 30ºC.
?. Day 2: Induction of the recombinationThe tubes were shaken overnight at 45ºC so as to induce FLP recombinase expression and select for the loss of pCP20.
?. Day 3: Plating to get single candidate recombinants100-fold dilution of the overnight culture was made by using fresh LB.50 ul of the diluted overnight culture was plated on LB plate.The plate was then incubated for 1.5 days under 30ºC to prevent partial loss of plasmid from the colonies which developed from the cells containing pCP20.
?. Day 4: Screening for genomic recombination and plasmid loss6 colonies were picked from each plate and resuspended in MilliQLittle amount of the MilliQ with cells was streaked on the LB plate(without antibiotics). The remaining solution was divided over LB + AMP, LB + CHL and LB + KAN plates dropwise for screening.The plate was then incubated overnight under 37ºC.
?. Day 5: Obtaining successful recombinants.
TITLE: Sedimentation Assay
AUTHORS: Michael X. Henderon

[DESCRIPTION]
This protocol details sedimentation assay for alpha-synuclein fibrils.

[STEPS]
SECTION: With Sucrose Cushion
1. 
 

Dilute 4 µL of 5 mg/mL α-synuclein PFFs to 40 µL in PBS in ultracentrifuge tubes.

SECTION: With Sucrose Cushion
2. Add 40 µL 20% sucrose beneath PFFs.

SECTION: With Sucrose Cushion
3. Ultracentrifuge at 100000 x g (45000 rpm) for 30 min at 22 °C.

SECTION: With Sucrose Cushion
4. Remove 70 µL supernatant and add to new tube.

SECTION: With Sucrose Cushion
5. Add 60 µL 12.5% sucrose to the pellet and resuspend the pellet by pipetting.

SECTION: With Sucrose Cushion
6. Dilute samples in 5x sample buffer.
SECTION: With Sucrose Cushion
7. Boil samples at 95 °C for 5 min.

SECTION: With Sucrose Cushion
8. Run samples on a 15% polyacrylamide gel.

 

SECTION: With Sucrose Cushion
9. Stain with Coomassie blue.

SECTION: Without Sucrose Cushion
10. Dilute 4 µL of 5 mg/mL α-synuclein PFFs to 40 µL in PBS.

SECTION: Without Sucrose Cushion
11. Ultracentrifuge at 100000 x g  (45000 rpm) for 30 min at 25 °C.

SECTION: Without Sucrose Cushion
12. Remove supernatant and dilute in 5x sample buffer.
SECTION: Without Sucrose Cushion
13. Add 40 µL PBS to the pellet.

SECTION: Without Sucrose Cushion
14. Resuspend pellet by pipetting up and down. Dilute in 5x sample buffer.
SECTION: Without Sucrose Cushion
15. Boil samples at 95 °C for 5 min.

SECTION: Without Sucrose Cushion
16. Run samples on a 15% polyacrylamide gel.

 

SECTION: Without Sucrose Cushion
17. Stain with Coomassie blue.