eamag/protocols-with-steps huggingface dataset:
TITLE: A protocol of molecular detection of phytoplasmas and Xylella spp. in post-entry quarantine for plants.
AUTHORS: Takao Ito, Ryoji Nakaune

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In the STEPS, we describe TaqMan multiplex real-time PCR to universally detect phytoplasmas (PP) and Xylella spp. (XL) with plant internal control (IC) from crude extracts. A protocol file [Protocol-JpEnXX.pdf] shows further details of the protocol in Japanese and English.</div></div>

[STEPS]
?. 1.　Extraction1.1.　Crude extraction1.1.1.　Put leaf petioles (＜50mg), a metal beads, and 1mL extraction buffer into a tube.1.1.2.　2,500 rpm 60 sec. (the Multi-beads shocker)1.1.3.　9,000 x g 10min 4C1.1.4.　Transfer the supernatant to a new tube. Next steps, or keep it in a freezer.
?. 1.2. Isopropanol precipitation1.2.1.　Add an equal volume of cold isopropanol to the crude extract and mix.1.2.2.　20,000 x g 5 min 4C1.2.3.　Discard supernatants and dry pellets.1.2.4.　Suspend the pellet in one-fifth volume of TE. Next steps, or keep it in a freezer.
?. A12.　Real-time PCR
2.1.　Reagent mixture




Reagents


1 reaction


10 reactions




Sterile water


2.5


25




TaqMan FAST Advanced Master Mix


5


50




Primer mixture


1


10




Probe mixture


1


10




Total (µL)


9.5


95




2.2.　Dispense 9.5 µL of the reagent mixture to PCR tubes
2.3.　Add 0.5 µL of the extract (1.2.4) to the tube.
2.4.　Set the tubes and run the StepOnePlus with the following parameters:
50C 2 min. → 95C 20 sec. →
95C 1 sec. → 60C 20 sec. （50 cycles）
* Targets（Reporter/Quencher）：PP（FAM/NFQ-MGB）, XL（VIC/NFQ-MGB）, IC（TAMRA/None）

See the next step.
?. 3.　Data analysis3.1.　Export data3.2.　Consider positives of PP/XL at Ct3.3.　Refer the PDF file (a protocol in Japanese and English) in the Absract for detail.
TITLE: Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Skeletal Muscle
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics protocol to fix, dissociate, and profile RNA from human skeletal muscle tissue. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Cell/Nuclei Isolation Protocol for Human Skeletal Muscle
1. The protocol CG000553 REV B was used to fix, dissociate, and isolate cells/nuclei from frozen human skeletal muscle with the following modifications: 

1) 1 mg / mL of Liberase TH was used for dissociation at Step 2b, Page 6.
2) Two extra "spin only" (i.e., steps 3 and 4 ) of the Octodissociator protocol were run for each sample due to the presence of intact/ large tissue pieces at the end of the run at Step 2c, Page 6.   
3) Counts were performed using the Cellaca PLX Automated Cell counter (PN: PLX-SYS1) and ViaStain AOPI staining solution (PN: CS2-0106-5mL) at Step 2i on page 6. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/sample-prep/tissue-fixation-and-dissociation-for-chromium-single-cell-gene-expression-flex


SECTION: Chromium Fixed RNA Profiling Reagent Kits
2. The protocol CG000527 Rev E  was used to generate gene expression libraries from fixed cell/nuclei suspension inputs.

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples

Samples were multiplexed in batches of 16 using the user guide CG000565. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples
TITLE: HEK293T Landing Pad Recombination Protocol with Fugene (Based on a 24-well plate)
AUTHORS: Sarah Roelle, Kenneth Matreyek

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For using Fugene 6 to recombine Bxb1 attB plasmids into Bxb1 attP Landing Pads already integrated into HEK 293T cells.</div></div>

[STEPS]
?. [Day 0: Day of transfection]
Trypsinize and count the cells.
?. [Day 0: Day of transfection]
Make 2 transfection mixtures per sample,Opti1 and Opti2 (DNA volumes should be kept between 1.0 and 5.0 uL, if possible):
?. [Day 0: Day of transfection]
Opti1:   +
[Opti-MEM]
[Fugene6]
?. [Day 0: Day of transfection]
For the Bxb1-containing sample - Opti2:   + µL corresponding to   + µL corresponding to             ----OR----
[Opti-MEM]
[Bxb1 expression plasmid]
[attB plasmid]
?. [Day 0: Day of transfection]
For the No-Bxb1 sample- Opti2-noB:     + µL corresponding to
[Opti-MEM]
[attB plasmid]
?. [Day 0: Day of transfection]
Add   to each well.(Be sure media contains doxycycline to a final concentration of  .)
[media]
2
?. [Day 0: Day of transfection]
Plate 120,000 cells per well within a 24-well plate.
?. [Day 0: Day of transfection]
Once at least 15 minutes have past since the mixtures were completed, add them dropwise throughout the well.  Rock the plate back and forth to mix it around the well.
?. [Day 1: Adding more media to cells]
Add at least   to each well.
[media]
?. [Day 7+: Read out the recombination]
Run the samples through the flow cytometer.  Watch the cells on the days in between.  They may need to be split at some point.  It's probably easiest to simply plate all the cells into a 6-well plate when they need to be split.
?. [Day 3+: Negative and/or positive selection]
If using a landing pad that encodes iCasp9, then add AP1903 to a final concentration of  to induce apoptosis of un-recombined cells. Dying cells can be observed as soon as 2 hours after AP1903 addition, and can be removed by changing the media.
10
If you want to estimate how many cells were recombined (this is recommended, especially for library -based experiments), make sure you leave some cells WITHOUT selection, so you can see what percent of the cells are reporter (eg. mCherry) positive. Then, you can multiply that percentage with the total number of cells you recombined (eg. 120,000 cells if only recombining a single well) to get the total number of recombined cells.
?. [Day 3+: Negative and/or positive selection]
If using an attB vector encoding a positive selection marker, add the small molecule. For puromycin resistant recombination plasmids (this is most often the case), add puromycin to a final concentration of .
1
If performing both negative AND positive selection, we typically perform the negative selection first, and only perform the positive selection after the well has reached greater than 20% confluency.
TITLE: Volume of oils (fatty acids) in coffee and comparison between roast levels
AUTHORS: Shannon.logan

[DESCRIPTION]
Measuring the volume of oils produced in coffee across a range of roasts.

[STEPS]
1. Grind coffee to desired consistency and weigh 20g to put into cafetiere
2. Boil kettle with 250ml of deionised water
3. Pour boiling water into a beaker and allow to cool to 93 °C, using a digital thermometer to ensure accuracy of temperature.

4. Once the water has cooled to the desired temperature, pour into the cafetiere and allow to brew for 4 minutes, using a timer/stopclock.
5. Pour each 2 of each sample into the centrifuge tubes, and centrifuge samples at 6000rpm for 30 minutes.
6. When the centrifugation is complete, using a syringe, remove the top layer of oils from the sample, and measure against the syringe measurements. 
7. Take note of each sample's volume.
TITLE: RNA clean-up by phenol:chloroform
AUTHORS: Daniel Richter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Phenol: chloroform extraction to clean up RNA ( e.g. to remove DNAse or DNAse Inactivation Reagent.)

</div><div class = "text-block">Based on phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)</div></div>

[STEPS]
?. Add 40 μL (1/10 of final volume) of 3 M sodium acetate, pH 5.2.
Final concentration will be 0.3 M.
[3 M sodium acetate, pH 5.2]
?. Add 880 μL (2x volume) of phenol:chloroform:isoamyl alcohol pH 8.0
880 µl
?. Shake vigorously inside fume hood for 15 seconds.
[Shake]
?. Centrifuge at maximum speed in a microcentrifuge at 4° C, for 2 minutes.
[centrifuge]
?. Check solution for insoluble precipitate, indicating the presence of potassium salts > 20 mM.
?. Transfer 420 μL of the top aqueous phase to a new tube.
[Transferred into a new tube.]
?. Add 840 μL of chloroform:isoamyl alcohol pH 8.0
?. Shake vigorously inside fume hood for 15 seconds.
[Shake]
?. Centrifuge at max speed for 2 minutes at 4ºC.
[Centrifuge]
?. Transfer 400 µL of the top aqueous phase to a new tube.
?. Add 1.5 µL of GlycoBlue.
Final amount ~ 25 micrograms, working stock 15 micrograms/microliter.
[GlycoBlue]
?. Add 1000 µl (2.5 volumes) of 100% ethanol
1000 µl
?. Shake vigorously inside fume hood for 15 seconds.
[Shake]
?. Freeze overnight at -20°C.
?. Centrifuge at maximum speed for 10 minutes at 4°C.
[Centrifuge]
?. Pipette out ethanol, slowly, with P1000 then P10.
Avoid leaving liquid on sides of the tube.
?. Wash with -20 °C, 70% ethanol and centrifuge at maximum speed for 5 minutes at 4° C.
?. Pipette out ethanol, slowly, with P1000, then P10.
Avoid leaving liquid on sides of tube.
?. Dry pellet and resuspend in 27.5 µL of water.
[Water]
?. Spec on NanoDrop and dilute 1 µL aliquot of RNA to 1 ng/µL in a separate tube (for Bioanalyzer)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This revisited Thalassiosira pseudonana conjugation protocol builds on the following two protocols: <a href="https://www.protocols.io/view/conjugation-of-thalassiosira-pseudonana-f55bq86" target="_blank">Conjugation of Thalassiosira pseudonana</a> (DOI: dx.doi.org/10.17504/protocols.io.f55bq86) &amp; <a href="https://www.protocols.io/view/pour-plating-of-thalassiosira-pseudonana-tp-jfncjme" target="_blank">Pour plating of Thalassiosira pseudonana (Tp)</a> (DOI: dx.doi.org/10.17504/protocols.io.jfncjme). Readers are strongly encouraged to read both before using this one. Gene-EGFP fusion depicted in the 'Expected Results' section was expressed under consitutive Tp-derived fcp promoter and terminator using pTpPuc3 episome backbone described in Karas <em>et al. </em>(2015). </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: DNA Ligation
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for how to perform DNA ligation. To see the full abstract and other resources, visit </span><a href="https://www.addgene.org/protocols/dna-ligation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/dna-ligation/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration. However, for most standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend around  of total DNA in a standard ligation reaction. Use a ligation calculator to easily quantify how much vector and insert DNA to use.Combine the following in a PCR or Eppendorf tube:Vector DNAInsert DNALigase Buffer (/ reaction for 10X buffer, and / reaction for 5X buffer)- T4 DNA LigaseH2O to a total of
100 ng
1 µl
10 µl
2 µl
10 µl
0.5 µl
1 µl
10 µl
Notes: If the DNA concentrations are low such that you cannot get all   of DNA, buffer and ligase into a   reaction, scale the reaction size as necessary - being sure to increase the amount of buffer proportionally.   of ligase should be sufficient for larger ligation reactions.Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to take a fresh tube, thaw it one time and aliquot individual tubes of  ,   or   for storage at  . Whenever you need to set up ligations in the future you can thaw a new tube that you know has only been thawed once before.Always do controls. See the tips and general guidelines section for detailsTry different vector to insert ratios to optimize the ligation reaction. See the tips and general guidelines section for details.
?. Incubate at  for , or at  overnight (following the manufacturer’s instructions).
0 Room temperature
16 °C
Note, for many ligation reactions, especially if using "high concentration" ligase,   at   is enough. For trickier ligations (such as ligation of annealed oligos) the efficiency of ligation can be improved by incubation at  .
?. Proceed with bacterial transformation.
TITLE: A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks
AUTHORS: Sang Hyo Kim, Caleb  Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier  D. Monzón

[DESCRIPTION]
This protocol details a novel method to isolate individual ticks and manipulate their environment. We successfully used this method to investigate how humidity affects survival and host-seeking (questing) behavior of three species of ticks: the lone star tick (Amblyomma americanum), American dog tick (Dermacentor variabilis), and black-legged tick (Ixodes scapularis). We placed 72 adult females of each species into individual plastic tubes and separated them into three experimental relative humidity (RH) treatments representing distinct climates: 32% RH, 58% RH, and 84% RH. For 30 days we assessed the survival and questing behavior of each tick.

[BEFORE_START]
We acquired adult female ticks from the Oklahoma State University Tick Rearing Facility and experimented with one temperature range and three relative humidities. This protocol can be modified for different species, life stages, temperatures, humidities, and other small organisms. 

[GUIDELINES]
Questing qualifications: 
The individual is not walking but it is still with its front legs extended       

Death qualifications: 
If any tick appears dead, lightly blow on it since ticks respond to carbon dioxide exhaled by potential hosts
If the tick does not move at all in 2 minutes, it should be counted as dead and placed in 70% ethanol

[STEPS]
SECTION: Set up
1. Place a single tick with one wooden skewer in each tube and seal with a cap, labelling each tube with an individual identifier  
  
SECTION: Set up
2. Place six tubes in each airtight container along with a humidity pack, labelling each container
     

SECTION: Set up
2.1. Confirm the humidity in one container of each RH level with the data logger 

SECTION: Set up
3. Program the climate chamber
 

SECTION: Set up
3.1. To cycle between 20 °C to 30 °C, the temperature increments should be as follows: 
3:00  - 25 °C 
6:00  - 27.5 °C 
9:00  - 30 °C 
12:00  - 27.5 °C 
15:00  - 25 °C 
18:00  - 22.5 °C 
21:00  - 20 °C 
24:00  - 22.5 °C 


SECTION: Set up
3.2. To create a 12:12 light:dark photoperiod, lighting increments should be as follows:
9:00 - Lights on
21:00 - Lights off
SECTION: Data collection
4. Place all of the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program
SECTION: Data collection
5. Each day thereafter, during the 9:00 to 12:00 or 30 °C increment, assess each tick for survivorship and questing behavior (see guidelines for qualifications of survivorship and questing)
 

 


SECTION: Data collection
5.1. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers has occurred
SECTION: Data collection
6. Repeat steps 4 and 5 for 30 days or until all ticks have died
TITLE: Plant Extraction and Fractionation
AUTHORS: Matin Mahmood, Enas Jawad Kadhim, Abdulkareem Hameed Abd

[DESCRIPTION]
Plant extraction is a process that aims to extract certain components present in plants. It is a solid/liquid separation operation: a solid object (the plant) is placed in contact with a fluid (the solvent). The plant components of interest are then solubilized and contained within the solvent

[STEPS]
3. Powdered plant materials were defatted with hexane in ratio 1:3 W/V for 24 hr.( 500 gm of plant material with 1500 mL of hexane)  

4. Allowed to dry at room temperature
1. A (500gm) of shade-dried for 12 days.
2. Coarsely powdered plant materials
5. The defatted plant materials were extracted with (2 Liters) of 80% ethanol in soxhlet apparatus until complete exhaustion.
6. The alcoholic extract was evaporated to dryness, under reduced pressure at a temperature not exceeding 40 هC by using IKA RV 10 Rotary Evaporator to give a dark greenish-yellow residue designated as a crude fraction (A)
7. Crude fraction was acidified with 300ml of 5% hydrochloric acid to pH 2 
8. Crude fraction then partitioned (three times) with equal volume of ethyl acetate to get two layers (aqueous acidic and ethyl acetate layer), this step is necessary to get rid from any basic compound found in the crude extract.
9. The ethyl acetate layer of the original alcoholic extract (crude fraction) was evaporated to dryness under reduced pressure 
10. Basified with 300ml of 5% sodium hydroxide to pH 10 and extracted with chloroform in the separatory funnel to get two layers
11. The aqueous basic layer which was separated, evaporated to dryness and acidified with 5% HCL to pH 2 then extracted with ethyl acetate to get fraction designated as fraction 3 (F-B) 
12. The second layer-chloroform layer which was also separated and evaporated to dryness under reduced pressure then partitioned with 80% methanol and petroleum ether to get two layers petroleum ether fraction (C) and 80% methanol fraction which designated as fraction 4 (F-D)
2. 
2. 
SECTION: Protocol Scheme
13.
TITLE: Deglycosylation of N-glycosylated proteins using PNGase F 
AUTHORS: Kaia Kukk

[DESCRIPTION]
The described protocol was used to confirm that NADPH-cytochrome P450 reductase from Helianthus annuus was N-glycosylated when recombinantly expressed in Pichia pastoris (Komagataella phaffii).

[STEPS]
1. 12,5 µl of yeast lysate containing sufficient amount of target protein for detecting by Western analysis was pipetted into a tube. Two samples were prepared in parallel, one for negative control without N-glycosidase and the other with PNGase F.
2. 0,5 µl of 10% sodium dodecyl sulfate (SDS) and 1 µl of 1M dithiothreitol (DTT) were added.
3. The mixtures were incubated at 95 °C for 5 min.
4. The mixtures were cooled to room temperature after which 2 µl of 0,5 M Tris-HCl, pH 8 and 2 µl of 10% Triton X-100 were added.
5. 2 µl of PNGase F (500 U/ml) was added to one of the samples. Instead of N-glycosidase, water was added to the negative control sample.
6. The samples were incubated overnight at 37 °C.

7. SDS-PAGE sample buffer was added and the samples were heated for 5 min at 99 °C. SDS-PAGE and Western analysis were carried out to see whether the molecular weight of the target protein had decreased.
TITLE: Collecting samples for Total Organic Carbon (TOC) analysis
AUTHORS: Krista Longnecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes collecting samples for Total Organic Carbon (TOC) analysis. The method is appropriate for samples in oligotrophic marine waters where the concentration of particulate organic material is low.</div></div>

[STEPS]
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
The glass vials need to be combusted before use.
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Set aside the caps that were sent with the vials because they cannot be combusted.
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Wrap packs of glass vials in foil prior to combusting them.
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
In the field, while wearing gloves, rinse the 40 ml glass vials 3x with sample water.
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Fill each vial up to the shoulder with the sample ().
40 mL
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Add  of  hydrochloric acid to this vial.
40 µl
12
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Store the acidified samples at  in racks/boxes that keep vials from knocking into each other.
4 °C
One good idea is to process several MilliQ samples in the field the same way the water samples are processed to test for potential contamination in the field.
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Combust the glass vials at  for at least .
450 °C
?. [Collecting samples for Total Organic Carbon (TOC) analysis]
Use electrical tape or lab tape to label the vials. Do not use too much tape as that causes problems with inserting the vials into the autosampler. We prefer 1/2 inch label tape, placed towards the top of the vial. Be sure to wrap the tape all the way around the vial in order to prevent the labels from falling off during sample processing.
TITLE: BSCI:414--Lab10: Protein Translations and In Frame Insertions
AUTHORS: Harley King

[STEPS]
?. [Translate Spike-Sumo into Protein]
Find the plasmid "F20_spike-SUMO/pET28" in Benchling in the BSCI:414 plasmids root folder. Open it. Copy this to a new folder with your name under "BSCI:414 Lab 10."
?. [Translate Spike-Sumo into Protein]
Locate the first "ATG" codon after the ribosomal binding site (RBS), downstream of the T7 promoter.  Put the cursor to the left of base A. Now, locate the stop codon. While holding down shift, select the last base in the stop codon. NOTE: The stop codon can be difficult to find, so select the bases all the way up to the "T7 Terminator" sequence. You can adjust the translation after identifying the position of the stop codon. The sequence between the start codon and the stop codon should be selected and highlighted. Right click with your mouse pointer on the highlighted sequence and select "Create Translation>Forward."This is the sequence of the combined or chimeric protein Spike and SUMO and pET28.
?. [Translate Spike-Sumo into Protein]
Annotate the protein sequence when the correct translation, with a proper start and stop codon, have been identified. Again, ensure the sequence between the start and stop codons has been selected, right click on the DNA sequence, select "Create Annotation". In the name put "spike-sumo protein." In "Annotation type" coding sequence or "cds". Now it's easy to identify the chimeric protein sequence by selecting the annotation.
?. [Create Coding Sequence for an Unknown Protein]
Open a plasmid sequence you haven't seen before,  "F20_unknown_protein/pET28."
?. [Create Coding Sequence for an Unknown Protein]
Find and annotate the coding sequencing using the steps above.
?. [Create Coding Sequence for an Unknown Protein]
Select the annotation and right click in the sequence. Click "NCBI BLAST" to identify the sequence. 1) Update the annotation to reflect the protein's name.2) Rename the plasmid.
?. [Translate Spike-Sumo into Protein]
Find the polypeptide "QCVN" using CTRL- or COMMAND-F.
?. [Translate Spike-Sumo into Protein]
Place the cursor between 16V and 17N. Press "G" on your keyboard and watch what happens to the downstream sequence. At what position is the new stop codon?
?. [Translate Spike-Sumo into Protein]
Press "G" again on your keyboard. What happens to the downstream sequence?
?. [Translate Spike-Sumo into Protein]
Press "G" again on your keyboard. What observations can you make regarding insertions in a coding sequence?