eamag/protocols-with-steps huggingface dataset:
TITLE: High Efficiency Transformation 
AUTHORS: New England   Biolabs

[DESCRIPTION]
Protocol for high efficiency heat shock transformation of competent E. coli cells.
Source: New England Biolabs

[STEPS]
1. Thaw a tube of competent E. coli cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into a transformation tube on ice.

2. Add 1 µL containing 100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 min. Do not mix.

4. Heat shock at exactly 42 °C for exactly 30 s. Do not mix.

5. Place on ice for 5 min. Do not mix.

6. Pipette 950 µL of room temperature SOC into the mixture.

7. Place at 37 °C for 60 min. Shake vigorously ( ) or rotate.

8. Warm selection plates to 37 °C.

9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37 °C. Alternatively, incubate at 30 °C for 1440 min or 25 °C for 48 hours.
TITLE: PBS 1X
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recepe is used in the following protocols:</div><div class = "text-block">- Separation and purification of human PBMC from FRESH BLOOD</div><div class = "text-block">- Separation and purification of human PBMC from BUFFY COAT</div><div class = "text-block">- Magnetic bead-based CD4+ T cell isolation from PBMCs with Dynabeads: CD4 Positive Isolation Kit</div><div class = "text-block">- Magnetic bead-based TREG-TEFF cell isolation from PBMC with Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit</div><div class = "text-block">- Staining of human PBMC or ISOLATED SUBSETS with Cell Proliferation Dye-eFluor™ 670 (CPD-eFluor670) for cell proliferation evaluation by Flow Cytometry</div></div>

[STEPS]
TITLE: Thawing Frozen Adherent Cell Lines
AUTHORS: Carolina Lopez

[DESCRIPTION]
This protocol describes the procedures from thawing and expanding adherent cell lines

[STEPS]
SECTION: Protocol (Sydney Faber_2023)
1. Day 1
Prepare media ahead of time and warm to room temperature
Spray down each bottle with 70% Ethanol before placing it into the TC hood
Pre-cool the centrifuge to 4C
Prepare and label a 6-well plate, and aliquot 9mL of media into a 15mL conical for each tube of cells to be thawed
Find the location of the cells you will be thawing in the -80C freezer inventory google doc
Transfer the cells between the freezer and the TC room on ice
Record the information on the cryotube – date, number of cell, etc
Begin adding media from the 15mL conical to the still frozen cells, then transfer media back and forth between the two tubes until the cells have completely thawed and everything has been transferred to the 15ml conical
Spin down the cells at 1200rpm for 10’ at 4C 
Pour off the supernatants and break up the cell pellets by tapping the tubes
Add 1mL of media per 2x10^6 cells to the cells, pipette up and down to break up any clumps, and transfer to the 6-well plate
Add 10ul of Mycoplasm removal agent per 1mL of media
Confirm the presence of cells in the well by microscope
Incubate at 37C
SECTION: Protocol (Sydney Faber_2023)
2. Day 3 *If cells tested negative for mycoplasma before freezing, only 1 round of MRA treatment is necessary. If mycoplasma presence is questionable or unknown 2 rounds of MRA treatment should be done – see mycoplasma protocol for more information.
Warm media to room temperature
Remove media and wash cells once with PBS
Add 1mL of 0.05% Trypsin-EDTA to wells and incubate plate at 37C for 1-3 minutes until cells are completely released from the plate
Add 4mL of media to wells to halt trypsinization and transfer to a T25 flask
Incubate at 37C
SECTION: Protocol (Sydney Faber_2023)
3. Day 4
Warm media to room temperature
Check for cell adherence
If there are a lot of dead cells, change the media on the cells
If cells are nearing 50% confluence, trypsinize, transfer in 10mL to a T75 flask – Label as passage #0
TITLE: Yale - Creatine Kinase Activity
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the creatine kinase activity in blood , serum, and plasma. Creatine kinase activity is measured by the enzymatically coupled reactions of creatine kinase, hexokinase, and glucose-6-P dehydrogenase. The rate of NADPH formation is monitored by the change in absorbance at 340 nm. </div></div>

[STEPS]
?. Calibrate Cobas for the measurement of creatine kinase activity analysis by running two control serum.
?. Sample handling as performed by the Cobas Mira Plus.    a) Pipette 4.5 µL of sample into a cuvette slot.    b) Add 175 µL of CK NADP Imidazole Reagent.    c) Mixture is incubated at 37ºC and spun for 10 minutes.    d) Absorbance is measured at 340nm.
TITLE: Cardio Clear 7: Heart-Related Issues are Treated Quickly
AUTHORS: shjfgkoza  

[DESCRIPTION]
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[STEPS]
TITLE: Sequence processing and assembly workflow using CLC workbench, SortMeRNA, and MegaHit. 
AUTHORS: Helena Pound, Steven Wilhelm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol details one of many methods available to process and assemble sequence data using CLC workbench, SortMeRNA, and MegaHit.</div></div>

[STEPS]
?. Upload sequence files to CLC workbench, indicating whether the reads are paired-end or single-end. Choose quality control parameters.
?. We recommend removing failed reads and not demultiplexing.
?. Trimmed reads should then be exported as fastq files, maintaining 2 files for pair-end reads.
?. If multiple lanes were run for a single sample and they have not yet been interleaved, now is the best time to create a new sequence list that contains all sequencing data from multiple lanes/runs for a single sample.
?. Assemble "clean" sequence files using MegaHit version 1.2.9. The clean files should be named sample_notaligned.fasta.
?. This example uses the interleaved paired-end "clean" sequence files generated by SortMeRNA. Additional assembly options for other sequence types and stringency parameters are available. The final assembly will be labeled final.contigs.fa in the assembly_folder indicated.
?. Kopylova E., Noé L. and Touzet H., "SortMeRNA: Fast and accurate  filtering of ribosomal RNAs in metatranscriptomic data", Bioinformatics (2012), doi: 10.1093/bioinformatics/bts611.
?. The example is for paired reads, denoted by the R1 and R2 read files and the -paired_in function. Read files can be compressed with .gz or uncompressed. Note that each run must have a new working directory or it will overwrite existing files. This is particularly important if you are running multiple terminals of SortMeRNA at once. The output file denoted as sample_notaligned.fasta is the remaining interleaved, pair-end "clean" sequences you will use to assemble.
?. Remove any residual rRNA from sequences using SortMeRNA version 4. Note, this is not necessary if you have sequenced DNA. We recommend using all 8 databases provided. See example code below.
?. We recommend using a quality score of 0.02 (the lower the score, the more stringent) and using ambiguous trimming with a limit of 2. Read length parameters varies based on the length of reads you requested from the sequencer. Adapter removal can either performed automatically or by uploading adapter list provided by sequencer.
?. Trim sequences and remove adapters in CLC.
?. Li, D., Luo, R., Liu, C.M., Leung, C.M., Ting, H.F., Sadakane, K.,  Yamashita, H. and Lam, T.W. MEGAHIT v1.0: A Fast and Scalable Metagenome Assembler driven by Advanced Methodologies and Community  Practices. Methods (2016).
TITLE: Isolation of Extracellular Vesicles from Cell Culture Media by Differential Ultracentrifugation 
AUTHORS: Dima Ter-Ovanesyan, Wendy Trieu , Maia Norman, Roey Lazarovits, George Church, David Walt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extracellular vesicles (EVs) are released by all mammalian cells and are thought to be important mediators of</div><div class = "text-block">intercellular communication. There are many methods for isolating EVs from cell culture media, but one of the</div><div class = "text-block">most commonly used methods continues to be purification based on ultracentrifugation. This approach has the advantage of allowing a large input volume of cell culture media. Here, we provide a detailed</div><div class = "text-block">protocol for isolating EVs by differential ultracentrifugation.</div></div>

[STEPS]
?. Culture cells under standard conditions to 50–70% confluency.
?. [Day 1]
For adherent cells: Aspirate media from twelve 15 cm plates.
?. [Day 2]
After 24 hours, take off all media and divide among 50 mL falcon tubes.
?. [Day 2]
Spin at 300 x g for 10 minutes at RT to pellet the cells.
?. [Day 2]
Transfer supernatant to new 50 mL tubes leaving cell pellet behind.
?. [Day 2]
Spin at 2000 x g for 10 minutes at RT (to pellet any dead cells).
?. [Day 2]
Transfer supernatant to new 50 mL tubes leaving cell pellet behind.
?. [Day 2]
Spin supernatant at 16,500 x g for 20 minutes at 4 °C  (to pellet large EVs).
?. [Day 2]
Transfer supernatant to new 50 mL tubes, leaving pellet behind.
?. [Day 2]
Pass supernatant through Steriflip 0.22 μm filter.
?. [Day 2]
Transfer supernatant to polyallomer ultracentrifuge tubes. Centrifuge at 120,000 x g  (26,500 RPM with SW32Ti rotor) for 70 minutes at 4 °C.
?. [Day 2]
Remove most of supernatant, leaving ~2 cm of media above pellet. Add 5 mL PBS to each tube. Vortex on medium speed for a few seconds. Fill to top of each tube with PBS.
?. [Day 2]
Again, centrifuge at 120,000 x g for 70 minutes at 4 °C.
?. [Day 2]
Aspirate all of supernatant with Pasteur pipet without touching bottom of tube where pellet is located.
?. [Day 2]
Resuspend pellet either in PBS or directly in the desired lysis buffer for western blot. If storing for later use, resuspend in HEPES buffer, and store in -80 °C.
?. [Day 1]
For suspension cells: spin down desired total number of cells in six Falcon tubes at 300 x g for 5 minutes.
?. [Day 1]
Aspirate media and resuspend each cell pellet in 40 mL FBS depleted media or media made without FBS. Transfer contents of each Falcon tube to T75 flask and return to incubator.
?. [Day 1]
Add 20 mL FBS-depleted media or media made without FBS per plate and return cells to incubator.
TITLE: Preparation of ventral midbrain cells for transplantation
AUTHORS: Tyra Fraser, Lachlan Thompson

[DESCRIPTION]
This protocol outlines the preparation of iPSC derived ventral midbrain progenitors for xenotransplantation.

[BEFORE_START]
D19 iPSC derived ventral midbrain progenitors are used in this protocol


[GUIDELINES]
All work is to be conducted in well sterilised laminar flow hoods designated for human iPSC work where possible to minimise contamination

[STEPS]
SECTION: Experimental details
1.  Wash wells with 1 x PBS -/- 
Incubate cells in 484 µL of Accutase per cm2 at 37 °C. Note: D19 typically dissociate between 10-20mins.
Prepare a 15 mL falcon tube for cells. 
Check dissociation after 6 min by using a p1000 to pipette up and down twice onto the
cells. Observe if cells dislodge and are present in small clumps of 5-10 cells.
 If YES: Collect all cells into a 15 mL tube. Wash wells with Accutase to ensure all
cells are collected.
If NO:  Incubate plate for intervals of 2 min Pipette suspension up and down twice targeting
the large clumps/sheets at each interval to promote dissociation. Repeat until cells are dissociated into small clumps when you can transfer to a 15 mL tube. 
SECTION: Experimental details
2. Add Ri (1:1000)
Incubate the tube for 1 min intervals until cells are broken into mostly single cells/small clumps (~5cells).
Cancel the reaction with PBS -/- at a 1:1 dilution to Accutase.
Spin cells (300 x g, 3 min, 4 °C)
SECTION: Experimental details
3. Aspirate supernatant. Flick pellet twice.
 Resuspend in NBB27 + All (~2ml per 48well) + Ri 1:1000. 
Resuspend suspension and pipette 10 µL into a small Eppendorf tube for cell counting. Repeat
for a second Eppendorf tube.
Take 2 tubes and add 10 µL trypan blue to cells in each tube. 
Transfer 10 µL of mixed cells in haemocytometer.
Count cells in each quadrant.
Calculate total number of cells. Repeat this for tube 2 to ensure accurate cell counts. 

Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4

8.  Calculate the total volume needed to resuspend cells to a final density (typically between 100-150K/µl)and the amount of Ri 1:1000 required.
9.  Spin cells (300 x g, 3 min, 4 °C)
SECTION: Experimental details
4. Aspirate supernatant.
Add half the required media on top of the cells gently (i.e. if you have 2 million cells total final volume is 20 µLto achieve 100 000 cells/µL so add 10 µLof media (NBB27 + All + Ri 1:1000) to pellet. 
Using a P20 gently disturb the pellet in a circular motion, taking care not to damage cells by hitting the edge of the tube with the pipette tip. 
Once mixed, take up the suspension once or twice and transfer to a small Eppendorf tube. 
Measure the volume of cell suspension.
Add the appropriate volume of NBB27 + All + Ri 1:1000 required to reach the final volume. 
Place cells on ice and transfer to surgical room immediately for transplantation.
TITLE: Freezing protocol for Escherichia coli
AUTHORS: Andreas Sagen

[DESCRIPTION]
An optimized freezing protocol of E. coli based on Zimmer and Verrinder Gibbins 1997.

[STEPS]
SECTION: Preparation of LB Freezing Buffer
6. Fill 80 mL LB-Lennox to a Duran bottle
SECTION: Freezing procedure
11. Transfer a saturated culture of E. coli cells

 

SECTION: Preparation of LB Freezing Buffer
10. Fill to 100 mL with LB-Lennox
SECTION: Preparation of LB Freezing Buffer
9. Adjust pH to pH 7.0
SECTION: Preparation of LB Freezing Buffer
8. Dissolve salts by stirring
SECTION: Preparation of LB Freezing Buffer
7. Add 1.32 mL Potassium phosphate monobasic (1 Molarity (M)), 3.6 mL Potassium phosphate dibasic (1 Molarity (M)), 50 µL magnesium sulfate (1 Molarity (M)), 170 µL sodium citrate (1 Molarity (M)), 680 µL ammonium sulfate (1 Molarity (M)) and 4.4 mL glycerol.

 

Materials:
 
 
 
 
 
 
SECTION: Preparation of LB-Lennox
1. Add 800 mL distilled water to a Duran flask
SECTION: Preparation of LB-Lennox
5. Sterilize by autoclave at 121 °C for 15 min
SECTION: Preparation of LB-Lennox
4. Fill distilled water to a total of 1000 mL
SECTION: Preparation of LB-Lennox
3. Adjust pH to pH 7.0 with sodium hydroxide

SECTION: Preparation of LB-Lennox
2. Measure 10 g tryptone, 5 g yeast extract and 5 g sodium chloride

Materials:
 
 
 
SECTION: Freezing procedure
12. Centrifuge at 1000 rcf, 5 min
SECTION: Freezing procedure
13. Remove supernatant, and resuspend pellet in an 10% the initial volume
SECTION: Freezing procedure
14. Aliquot saturated culture into an appropriate container. For example:
    100 µL saturated culture in a 200 µL reaction tube
    1000 µL saturated culture in a 1.5 mL reaction tube
    1500 µL saturated culture in a 2.0 mL cryotube
SECTION: Freezing procedure
15. Transfer to an ultra-low temperature or vapor-phase nitrogen freezer for long-term storage
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Lminex Milliplex Soluble Cytokine Receptor 13-plex manufacturer's protocol</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Silver staining of a acrylamide gel. </p>

[GUIDELINES]
<p><span style="text-decoration: underline;"><strong>Fixate-solution:</strong></span></p>
<p>50 ml. ethanol </p>
<p>12 ml. acetic acid</p>
<p>50 µl filtered 37% formaldehyde</p>
<p> -&gt; 100 ml. H<sub>2</sub>O</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Thiosulfate-Stock:</strong></span></p>
<p>20 mg/ml Sodium Thiosulfate</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Pre-treatment solution:</strong></span></p>
<p>1 ml Thiosulfate-Stock:</p>
<p> -&gt; 100ml H<sub>2</sub>O</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Silver nitrate solution</strong></span></p>
<p>0.1 g silver nitrate</p>
<p>37.5µl filtered 37% formaldehyde</p>
<p> -&gt; 50ml H<sub>2</sub>O</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Developer solution:</strong></span></p>
<p>100µl filtered 37% formaldehyde</p>
<p>40µl Thiosulfate-Stock:</p>
<p>-&gt; 200ml H<sub>2</sub>O</p>
<p>12g. Sodium carbonate</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Stop-solution</strong></span></p>
<p>12ml acetic acid</p>
<p> -&gt; 100ml H<sub>2</sub>O</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
?. 
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?. 
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TITLE: E. coli CRISPR Barcoding
AUTHORS: Leanne Hobbs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the protocol for barcoding </span><span style = "font-style:italic;">E. coli</span><span> using CRISPR for CellRepo</span></div><br/><br/></div>

[STEPS]
?. [Day 1]
Start an overnight culture (37 °C) by inoculating LB medium from a single colony.
?. [Day 2]
Prepare competent cells following your favorite protocol.
?. [Day 2]
Transform E. coli cells with plasmid pREDCas9 and plate the cells at 30 °C in LB supplemented with 50 µg/mL spectinomycin.
?. [Day 3]
Start an overnight culture at 30 °C in LB/Spec from a single colony.
?. [Day 4]
Next morning refresh the culture with LB/Spec and grow the cells until OD600 reaches 0.1.
?. [Day 4]
Add IPTG to a final concentration of 2 mM and grow the cells to an OD600 =0.6 (recombination proteins are being expressed at this point).
?. [Day 4]
Frost cells on ice for 20 minutes and electrocompetent cells are then prepared by washing bacteria with ice-cold milliQ water after spinning aliquots 10 minutes at 5000 rpm in a 4 °C centrifuge. (Heat shock transformation also could be performed)
?. [Day 4]
After two washes, resuspend cells in the residual milliQ water and electroporate with pEC-CRISPR2-BC.
?. [Day 4]
Add 950 μl of fresh LB without antibiotics and resuspend cells. Incubate cells 1 hour at 30 °C.
?. [Day 4]
Then, spread 100 μL of bacterial culture on LB plates supplemented with Spec (50 µg/mL) /Amp (100 µg/mL).
?. [Day 5]
Check colonies for barcode presence by colony-PCR.
?. [Day 5]
Inoculate a positive clone in 2 mL of LB/Spec/Ara (30 mM) (sgRNA targeting pUC origin in pEC-CRISPR2-BC is expressed).
?. [Day 5]
Grow for 4-6 hours.
?. [Day 5]
Plate on LB/Spec.
?. [Day 6]
Check some colonies for Ampicillin resistance by restreaking them on LB/Amp.
?. [Day 7]
Take a sensitive clone and restreak it on LB plates at 37°C.
?. [Day 8]
Check again by colony PCR the presence of the barcode and restreak on LB/Spec and LB/Amp plates to double check plasmid curing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to extract total RNA from flatworms. It is from:<br /><br /><em><span style="color: #222222; font-family: arial, sans-serif; font-size: 12.8px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: #ffffff;">Hebert, F, O; Grambauer, S; Barber, I; Landry, C, R; Aubin-Horth, N (2016): Reference transcriptome sequence resource for the study of the Cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience Database. </span><a href="http://dx.doi.org/10.5524/100197" target="_blank">http://dx.doi.org/10.5524/<wbr />100197</a></em>

[BEFORE_START]
Pre-heat incubator at 65°C, clean bench surface and all the lab tools that will be used in the protocol (pipettes, tube holders, dissection forceps) with RNAse Zap/Rnase Wipes. Make sure that isopropanol + ethanol are at -20°C.

[STEPS]
?. 
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TITLE: Chapter 9: Release
AUTHORS: Kerri Wolter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes when, where, and how to release vultures after successful rehabilitation.</div></div>

[STEPS]
?. When releasing a vulture, lower your body (see CHAPTER 1 for pictures), bringing the bird’s feet to ground level, slowly allowing the bird to stand.
?. Then release your grip on its entire body and neck at the same time, stepping away to give the bird some space. Be careful never to drop or throw the bird down, nor allow the bird to fall prior to lowering it gently to ground level.
?. When releasing the bird or placing it back inside the enclosure, remain still and allow the bird to walk or fly away from you, rather than panicking it, in which case it may take off, flying into stationary objects / fences.
?. Do not force the bird to move. Allow the bird time to recover but monitor it for any unusual behaviour that could be a sign of heat exhaustion or injury from handling. The bird will decide for itself what to do next; it might fly off, run or drink water. Never force the bird to move or fly after the handling. Simply monitor and interfere only as a last resort if the bird appears not to be fit for release.
?. Vultures, when given the opportunity, will time their take-off to coincide with a thermal or an increase in wind strength, making the take-off easier. Birds in general prefer to take-off against the wind. Consider these factors when choosing the release site and direction.
?. Release a bird from a crate at ground level, and not from an elevated site such as the back of a vehicle.
?. Do not pull a bird out by a wing or the tail or tilt a crate to encourage a bird to exit. Give the bird time to leave the crate of its own accord.