eamag/protocols-with-steps huggingface dataset:
TITLE:     Culturing Physcomitrella patens
AUTHORS: Igem Dusseldorf

[STEPS]
?. [Execution]
Scrape 7 – 10 days old moss protonemal tissue with spatula.
?. [Execution]
Put 4 mL of water into a (conical tube). Put in the protonemal tissue. Cut it with a Homogenizer until no big chunk is visible and the water is uniformly green.
?. [Execution]
Spread 2-3 mL of freshly fragmented protonema on the cellophane on the petri dish.
?. [Execution]
Seal off with micropore tape to allow gas exchange.
?. [Execution]
Incubate plates at 24°C with a light regime of 16 h light : 8 h darkness at 80 μmol×m–2×s–1
?. [Execution]
For next harvest gently scrape surface of cellophane disks after 7 – 10 days (before culture starts to turn brown). Repeat cycle. PPNH4 liquid medium with 300 mg/L Cefotaxim or 250 mg/L Vancomycin is recommended for storage as the moss stays available for culture for >6 months.
?. [Prepare in advance]
Solid PPNH4 medium on sterile petri dishes
?. [Prepare in advance]
Cellophane disks with the appropriate size for the petri dishes. Wet the cellophane diskswith sterile ultrapure water to avoid creases.
?. [Prepare in advance]
Overlay the solid medium on the petri dishes with fitting cellophane disks to preventthe mosses from growing into the medium.
?. [Prepare in advance]
Liquid PPNH4 medium with 300 mg/L Cefotaxim or 250 mg/L Vancomycin.
?. [Prepare in advance]
Add 10 mL of a macro element-solution, 1 mL of a micro element solution, 1 mL of thepotassium phosphate solution and 2,7 mL of the ammonium tartrate solution for each liter ofmedium.
?. [Prepare in advance]
Add 7,2 g agar per liter of PPNH4-liquid medium. Add further 300 mg cefotaxime or 250 mg Vancomycin per liter ofmedium to suppress bacterial growth.
TITLE: Microbial Genome Editing
AUTHORS: contact.microbialtec  

[DESCRIPTION]
Early microbial genome editing mainly focused on random screening and simple rational screening, but the shortcomings of traditional methods that are time-consuming, labor-intensive, work-intensive, and non-directional mutagenesis results have gradually been exposed over time. With the development of modern molecular biology technology, there are many new methods of microbial breeding with more directional and positive mutation, which greatly promotes the accuracy and efficiency of microbial genome editing.

[STEPS]
1. Microbial Genome Editing Based on Rec

Microbial genome editing based on homologous recombination uses its own Rec system to carry out homologous recombination of exogenous DNA to achieve allelic replacement of target genes and recombination of genetic information between two homologous strands of DNA. By producing and isolating DNA fragments with genomic sequences similar to the part of the genome to be edited, these fragments are injected into monocytes. Once these fragments enter the cell, they can be recombined with the cell's DNA to replace the target part of the genome.

 

2. Microbial Genome Editing Based on CRISPR/Cas9

Microbes in nature always use various protection mechanisms that enable them to coexist with harsh environments and invasive nucleic acids. Many microbes can resist nucleic acid invasion through specific methods based on gene sequence. CRISPR/Cas9 is one of these protection mechanisms and the most widely used genome editing technology. The CRISPR/Cas9 method allows microbial genome editing such as gene knockout, site-directed mutation, site-directed insertion of foreign sequences, etc. Compared with traditional gene knockout methods, this method is more convenient for subsequent experimental research.

 
Fig.1 Mechanism of CRISPR/Cas9 action. Arora Let al. 2017)
TITLE: Phenol-chloroform DNA extraction from Sporosarcina pasteurii
AUTHORS: Michael S. Carter, Matthew J. Tuttle, Maneesh K. Gupta

[DESCRIPTION]
This protocol is for extraction of genomic DNA from Sporosarcina pasteurii. It is based on a standard phenol-chloroform DNA extraction method.

[GUIDELINES]
All steps should be performed in a chemical fume hood.

[STEPS]
SECTION: DNA extraction
1. Grow Sporosarcina pasteurii cells in 150 mL of BHI/330 mM urea at 30°C with 200 rpm shaking to an OD600 of 3.5.
SECTION: DNA extraction
2. Concentrate cells by centrifugation at 15,000 x g for 10 min and pour off the supernatant.
SECTION: DNA extraction
3. Resuspend cells in 10 mL of Resuspension Buffer (50 mM Tris pH 8.0, 10% sucrose).
SECTION: DNA extraction
4. Add 2.5 mL Lysis Buffer (Tris pH 8.0, 10 mg/mL lysozyme), 1.5 mL 10% SDS, 5 µL 100 mg/mL RNase A, and 25 µL 50 mg/mL proteinase K to lyse cells and stabilize DNA.
SECTION: DNA extraction
5. Incubate for 1 h at 37°C.
SECTION: DNA extraction
6. Add 1.3 mL of 3.0 M sodium acetate (pH 5.5) and 30 mL of 100% ethanol to separate DNA from other biomacromolecules by precipitation.
SECTION: DNA extraction
7. Spool DNA onto a glass hook and transfer to 20 mL of TE buffer.
SECTION: DNA extraction
8. Dissolve DNA in TE buffer for 1 h at 37°C.
SECTION: DNA extraction
9. Add a 5 mL aliquot of DNA to 7 mL of phenol:chloroform:isoamyl alcohol (25:24:1, Saturated with 10mM Tris, pH 8.0, 1mM EDTA) and mix by inversion.
SECTION: DNA extraction
10. Separate phases by centrifugation at 10,000 x g for 10 min.
SECTION: DNA extraction
11. Pipette off the aqueous phase and add to 5 mL of chloroform. Mix by vortexing.
SECTION: DNA extraction
12. Separate phases by centrifugation at 10,000 x g for 10 min.
SECTION: DNA extraction
13. Pipette off the aqueous phase and add to 30 mL of 100 % ethanol. Mix by inversion.
SECTION: DNA extraction
14. Collect precipitated DNA by centrifugation at 10,000 x g for 10 min.
SECTION: DNA extraction
15. Wash pelleted DNA twice by adding 5 mL of 70% ethanol, mixing by inversion, centrifuging at 10,000 x g for 5 min, and removing the supernatant.
SECTION: DNA extraction
16. Allow the pellet to dry.
SECTION: DNA extraction
17. Dissolve the final pellet in 500 µL of TE buffer at 37°C for 1 h.
TITLE: Model Building of PI3KC3-C1/RAB1A
AUTHORS: Annan SI Cook

[DESCRIPTION]
Details model building of the PI3KC3-C1/RAB!A complex using chimeraX1.5-1.6 and ISOLDE.  Refinement was done using Phenix real space refinement.

[STEPS]
SECTION: Generate Starting model
1. Use Alphafold2 to generate a starting model containing the PI3KC3-C1 components (VPS34, VPS15, ATG14, BECN1) and RAB1A respectively.  
SECTION: Docking and model building/refinement
2. Dock each starting model into the experimental map

SECTION: Docking and model building/refinement
3. Use ISOLDE to relax the entire PI3KC3-C1 complex and RAB1A into the experimental data in ChimeraX, selectively using restraints and ignoring clashes until the model simulates correctly. Trim obvious disordered regions by deleting them in Chimera.
SECTION: Docking and model building/refinement
5. Iteratively refine the model by building sidechains into density features, and improving the Ramachandran angles with selective restraints. 
SECTION: Docking and model building/refinement
6. Assess the model to map fit using the built in Q-score function and Ramachandran plot
SECTION: Docking and model building/refinement
7. When model fit to density and Ramachandran angles start to look good, export the model and refine using Phenix real space refinement. 
SECTION: Docking and model building/refinement
4. Build ligands into the map, use the ignore command to ignore clashes selectively until the clashes have been eliminated.
SECTION: Docking and model building/refinement
8. Repeat steps 5-7 until you are satisfied with the final model and the metrics cease improvement.
TITLE: Preparing 1% Eosin Y stock solution
AUTHORS: joshdm

[DESCRIPTION]
Preparation of 1% stock solution of Eosin Y for routine H&E staining. 

[STEPS]
1. Weigh 2g of Eosin Y disodium salt and place it into a 1000mL Earlenmyer flask
2. Measure 40mL of dionized water and add it to the flask. Swirl to dissolve Eosin powder. 
3. Measure 160mL of 95% ethanol and add it to the flask. Swirl to dissolve. 
4. Using a p1000 Pipette, add 1mL (1000uL) of Glacial Acetic acid to the solution. Swirl until homogeneous.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Simple 5-minute experiment to demonstrate volume of air to children.

[STEPS]
SECTION: Preparation
?. 
SECTION: Preparation
?. 
SECTION: Preparation
?. 
SECTION: Preparation
?. 
SECTION: Preparation
?. 
SECTION: Experiment
?. 
SECTION: Experiment
?. 
SECTION: Experiment
?. 
SECTION: Experiment
?.
TITLE: Verification guidelines for the DDNS method for poliovirus direct detection
AUTHORS: Joyce Akello, Alex Shaw, Catherine Troman, Erika Bujaki, Manasi Majumdar, Javier Martin, Nick Grassly

[DESCRIPTION]
This document describes a pragmatic approach to conduct in-house verification of the DDNS method for detection of polioviruses.  A laboratory should conduct an in-house verification on the entire DDNS method to confirm that the required performance characteristics can be met within the settings. This can prove that the laboratory can adequately perform the method as intended achieving the DDNS performance requirements for the sample matrix (stool) to which the DDNS method is applied for. Verification of the DDNS method will be assessed based on the following parameters:  accuracy and  sensitivity 

To verify the DDNS method performance, the laboratory should ensure that the lab personnel are adequately trained, competent,
and qualified to perform the DDNS method. It is the responsibility of each laboratory to ensure that staff have attended DDNS training,  biosafety risk assessment is performed, and adequate protective equipment is available to staff.

Note: We recommend working closely with members of the Polio Sequencing Consortium (PSC) before and during the verification of the DDNS method to ensure that the laboratory has all the required components and competency to perform the full protocol.

[STEPS]
TITLE: Coral DNA Extraction - Modified DNeasy PowerSoil Pro Kit
AUTHORS: Luigi   Colin

[DESCRIPTION]
DNeasy PowerSoil Pro Kit modified extraction protocol to improve yield with inhibitor heavy hard corals. 
Tested and developed on Acropora corals. Optimised for all coral DNA extraction that with high quantity of inhibitors

(Step 1 to 8 modified protocol; step 9 to 18 manufacturer’s protocol.)

[BEFORE_START]
Sterilise all the equipment that is not single use.
Ensure that the sample are as dry as possible before starting the protocol, taking particular care of removing any buffer they may have been kept in (Especially if RNALater was used)

[STEPS]
SECTION: Sample preparation
1. Gently crush a small piece of coral in a mortar and pestle (~100 mg, in order not to overload the column though). And transfer in a PowerBead Pro Tube (or 2 ml Microcentrifuge).
Note: For improved yield focus on adding more tissue and less carbonate from the skeleton, a scalpel 
SECTION: Sample preparation
2. Add 800 µL of buffer CD1 and vortex briefly.

SECTION: Cell lysis
3. Add 83 µL  of   and vortex briefly.
Note: quality of Proteinase K can be tweaked depending on the quality and type of samples 
SECTION: Inhibitor&#x27;s removals
5. Add 200 µL buffer CD2 and vortex for5 s

SECTION: Inhibitor&#x27;s removals
6. Microfuge a 16000 x g, 1 min .

SECTION: Inhibitor&#x27;s removals
7. Transfer supernatant to new tube (Max 700 µL).
 


SECTION: Bind DNA
8. Add600 µL of buffer CD3, vortex for 5 s

SECTION: Bind DNA
9. Load 650 µL of the lysate onto an MB Spin Column and centrifuge at 15000 x g, 1 min 

SECTION: Bind DNA
10. Discard the flow-through and repeat step 9 to ensure that all the lysate has passed through the MB Spin Column.
SECTION: Bind DNA
11. Carefully place the MB Spin Column into a clean 2 ml Collection Tube. Avoid splashing any flow-through onto the MB Spin Column.

SECTION: Wash
12. Add 500 µL of Solution EA to the MB Spin Column. Centrifuge at 15000 x g, 1 min

SECTION: Wash
13. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
SECTION: Wash
14. Add500 µL of Solution C5 to the MB Spin Column. Centrifuge at 15000 x g, 1 min

SECTION: Wash
15. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection
SECTION: Wash
16. Centrifuge at up to 16000 x g, 3 min. Carefully place the MB Spin Column into a new 1.5ml Elution Tube.

SECTION: Elute
17. Add 50 µL–100 µL of Solution C6 to the centre of the white filter membrane.

Note: To improve the yield use the lower volume and add a 10 min incubation at room temperature

SECTION: Elute
18. Centrifuge at 15000 x g, 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.

SECTION: Cell lysis
4. Incubate at 56 degrees for 60 minutes. Vortex for 10 s every 15 min (Ideally, do this on a shaking heat block, with shaking on full)

Note: If on orbital incubator at a minimum of 300rpm or more, can skip the vortexing.
Add an extra hour of incubation to increase yield. More than 120 min will results in shearing of the longer DNA fragments
TITLE: Cytotoxicity Assay Protocol
AUTHORS: Roshni Jaffery, Ningbo Zheng, Jiakai Hou, Ashley Guerrero, Si Chen, Chunyu Xu, Nicholas A. Egan, Ritu Bohat, Weiyi Peng

[DESCRIPTION]
This is the protocol for cytotoxicity assay in cancer cell lines using flow cytometry.

[STEPS]
1. Begin T cell culturing a week prior. Grow and culture tumor cell lines. Ensure the cells are not over-confluent. 
2. Split the tumor cells the day before the assay. Perform the cytotoxic assay as described in next steps.

3. Count tumor cells. Collect 3-5M cells for each cell line. 
4. Centrifuge the cells and wash the pellet once with PBS. Ensure that all media is washed off as this can inhibit the staining. 
5. Aspirate the supernatant and leave as little PBS as possible 
6. Re-suspend cells in culture medium at the appropriate ratio for the experiment (1X106 / ml for 50,000
cells/50ul/well or about ~80% confluent) using 96 well round well plate with 50,000 cell tumor and in triplication.
7. Add 50 ul of T cell suspension diluted to achieve the desired E:T ratio. Below is an example of how to set up E:T ratios:
 
 

8. Centrifuge the mixed cells in the tubes at 300 rpm for 5 minutes to gently pellet the cells. Incubate for 3 hours at 370C.
9. Add 150ul PBS into the plate and then centrifuge at 2000rpm for 5min.
10. Remove the supernatant and add then 100ul/well Trypsin. The plate is incubated at 37C incubator for 3-5mins.
11. Add 100ul FACS buffer to stop the trypsinization and centrifuge at 2000rpm for 5 min.
12. Remove the supernatant carefully.
13. Fix and permeabilize cells using Cytofix/CytopermTM Kit (BD BioSciences) for 20 minutes at room temperature (100ul/well – mix well).
14. Wash with Perm/WashTM Buffer (150ul/well) at 2000 rpm for 5 min.
15. Prepare caspase antibody dilution such that each well gets stained with 2.5ul of APC-conjugated rabbit anti-active caspase 3 Ab in 50 ul Perm/WashTM Buffer. Mix gently and incubate for 30 min in the dark on ice or in the 4°C refrigerator.
16. Wash cells 1Xwith Perm/WashTM Buffer (150ul).
17. Resuspend the cell pallets into 100ul FACS buffer (2% FBS in 1X PBS). 
18. Analyze the samples using flow cytometry.
TITLE: UC Davis - High fat diet feeding
AUTHORS: Kristin Evans

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary</span></div><div class = "text-block">A high-fat diet of varying composition and percent fat is administered to induce obesity in mice. High-fat diet induced obesity is causally associated with insulin resistance and type 2 diabetes.  Diets will be provided in consultation with the investigator needs.  This service applies to specialized feeding for vendor supplied animals being supplied out of the UC Davis vendor approved barrier facility.</div></div>

[STEPS]
?. High-fat diet is given ad libitum in mice.  Typically mice are started on the diet at 6 weeks of age (for C57BL/6 strains).  Age at diet start will be begin in consultation with the investigator needs
?. A stock bag of high-fat diet should be stored in a refrigerator.
?. High-fat diet placed in cages should be replaced 2 times a week to avoid spoilage.
?. Mice will typically eat more in the first 2 weeks of high fat diet feeding.  Careful attention to diet intake will be taken during this time.
?. Diet intake measurement and animal weights may be taken in consultation with the investigator as a separate service.
TITLE: PYG medium preparation (2 L)
AUTHORS: Carrie A Flynn, Barbara Kazmierczak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Recipe for PYG rich growth medium for </span><span style = "font-style:italic;">Acanthamoeba castellanii</span><span> trophozoites. Adapted from ATCC Medium: 712 PYG w/ Additives </span><a href="https://www.atcc.org/~/media/F7CFD291FAEC4B26AF234A3A9256C1A3.ashx" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.atcc.org/~/media/F7CFD291FAEC4B26AF234A3A9256C1A3.ashx</span></a></div></div>

[STEPS]
?. [Dry ingredients]
To a 2 L bottle, add:-stir bar
[sodium citrate dihydrate]
[yeast extract]
[bacto peptone]
?. [Water]
Bring volume to  with dH2O (for our reagents, requires adding  dH2O) and place on stir plate (unheated).
1.8 L
1.68 L
?. [Liquid stock solutions]
Add: 0.05 M CaCl2 x 2H2O 0.4 M MgSO4 x 7H2O 0.25 M Na2HPO4 (anhydrous) 0.25 M KH2PO4 (anhydrous) 0.005 M Fe(NH4)2(SO4)2 x 6H2O
16 mL
20 mL
20 mL
20 mL
20 mL
All of these nonsterile stock solutions can be made in advance in bulk and stored at room temperature.    0.05 M CaCl2 x 2H2O =   in   dH2O     0.4 M MgSO4 x 7H2O =   in   dH2O     0.25 M Na2HPO4 (anhydrous) =   in   dH2O     0.25 M KH2PO4 (anhydrous) =   in   dH2O     0.005 M Fe(NH4)2(SO4)2 x 6H2O =   in   dH2O
?. [Liquid stock solutions]
Stir until solution is clear. Check that pH is 6.5 with a pH strip.
?. [Liquid stock solutions]
Aseptically add  to the above medium.
[2 M glucose]
2 M glucose is made in advance, filter sterilized, and stored at 4ºC. This is made by slowly adding   to a beaker with approximately   while heating and stirring. Once all glucose is dissolved, bring final volume to   with dH2O and filter sterilize (0.22 µm).
?. [Sterilize]
Filter sterilize into sterile 250-mL glass bottles using 0.22 µm bottle-top filters.
?. [Sterilize]
Store at  for up to several months.
0 Room temperature
TITLE: Glycerol Stock Preparation for S. elongatus
AUTHORS: Akashdutta  

[DESCRIPTION]
Bacterial cultures in the form of agar plates or liquid cultures are not suitable for long term storage at low temperatures such as 4 degrees. Long-term storage of bacteria is best at temperatures around -80 degrees. However, agar plates and liquid cultures cannot be stored at such temperatures, as the water crystallizes in and around the bacterial cells, rupturing and killing them. Glycerol or DMSO (anti-freezing agents) is added to the media to store samples at such temperatures. 

[STEPS]
1. To make n stocks, take an aliquot of the S. elongatus culture such that the OD times the volume is at least 5n.
2. Centrifuge this aliquot in a falcon at 5000 rpm, 7 min, 25 °C 

3. Discard the supernatant.
4. Resuspend the pellet in the minimum amount of BG11 medium required to do so.
5. Add n mL of  25% glycerol and mix well. 
6. Separate equally into n cryovial tubes.
7. Store in the stocks in -80 °C .
TITLE: Entering the Lab (Covid-19 Shutdown Re-Entry, Srivastava Lab)
AUTHORS: Srivastava Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Disclaimer</span><span>: The SOP presented here has been designed by the Srivastava Lab at Harvard University and is being shared outside of Harvard for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
?. Hand sanitizer pump outside the door - use before unlocking door
?. Leave jacket outside of lab
?. Sanitize phone/any other object that will be in the workspace with 70% ethanol wipe
?. Wash hands at the sink
?. Wipe bench, pipettes, pens, etc with 70% ethanol
?. If it is your “high touch” day, wipe all handles (incubator, freezer, door, etc) with 70% ethanol
?. Turn on water
?. Wash hands
?. Dry hands with paper towel
?. Turn off faucet with paper towel
TITLE: Strand specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA
AUTHORS: Faizan Uddin, Madhulika Srivastava

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming reverse transcription reactions with gene specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA gets synthesized even without addition of exogenous primers in RT reactions. Due to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. The protocol described here was used for purification of biotin-tagged cDNA in conjunction with alkaline denaturation. This obviated the problem of background priming and enabled accurate strand specific detection of overlapping transcripts. </div></div>

[STEPS]
?. 2µg of DNase-I treated RNA was reverse transcribed at 55ᵒC using Superscript-III First-Strand Synthesis System for RT-PCR (Invitrogen 18068-015) in 40µl RT reaction following manufacturer’s instruction. The final concentration of each biotinylated GSP was 250nM.
?. 50µl of Dynabeads™ MyOne™ Streptavidin C1 were transferred to a 1.5ml tube and collected over the magnetic stand.
?. Beads were washed twice with 400µl of 1x binding buffer consisting of 1M NaCl, 1mM EDTA, 0.1% Tween-20 and 10mM Tris-Cl (pH 7.5).
?. Beads were resuspended in 400 µl of 1x binding buffer with gentle tapping and 35µl of RT reaction was mixed. Importantly, use of pipette was avoided for dispersion of the beads.
?. Tube containing beads and RT mix was incubated at 15ᵒC for 30 minutes with slow rotation for proper mixing.
?. Beads were collected by placing the tube over the magnetic stand for 2 minutes and supernatant containing unbound cDNA was aspirated out.
?. 200µl of 0.15M NaOH was then added to the bead pellet and beads were dispersed with gentle tapping, followed by an incubation of 10 minutes at room temperature with intermittent mixing to denature the cDNA-cDNA duplexes. Supernatant was removed using the magnetic stand.
?. Beads were washed once with 300µl of 0.1M NaOH to remove residual self-primed cDNA.
?. Beads were then washed twice with 400µl of 1x washing buffer consisting of 0.1M NaCl, 1mM EDTA, 0.1% Tween-20 and 10mM Tris-Cl (pH 7.5).
?. Finally, the beads were resuspended in 70µl of water and incubated at 70ᵒC for 5 minutes, followed by cooling on ice to release biotinylated cDNA.
?. A short spin at 1500g was given at 4ᵒC and enriched biotinylated cDNA was transferred to a fresh tube using the magnetic stand.
?. Enriched biotinylated cDNA (2 µl) was used for PCR. The amplification was carried out under conditions specifically optimized for each amplicon using gDNA and/or non-biotinylated cDNA as template.