eamag/protocols-with-steps huggingface dataset:
TITLE: Miracle Prep for Plasmid Isolation
AUTHORS: Blake Flood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Based on:</div><div class = "text-block"><a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160509" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160509</span></a></div></div>

[STEPS]
?. [Grow bacteria containing plasmid]
Allow to grow overnight at .
37 °C
?. [Isolate Plasmid]
Spin culture
Centrifuge: 4000 34
4 °C
?. [Isolate Plasmid]
Remove supt. Resuspend cells in  Buffer P1..
2 ml
?. [Isolate Plasmid]
Buffer P2 (invert 3-4 times)
2 ml
?. [Isolate Plasmid]
Incubate  @
37 Room temperature
?. [Isolate Plasmid]
Buffer N3 (invert 3-4 times)
2 ml
?. [Isolate Plasmid]
Distribute lysate into   1.5mL tubes
4
?. [Isolate Plasmid]
Centrifuge  @
Centrifuge: 13200 34
?. [Isolate Plasmid]
Collect supt, add 1 volume 96% EtOH, mix
?. [Isolate Plasmid]
Load on   “QIAprep 2.0 spin columns” or any generic silica membrane mini spin column (Econospin).
4
?. [Isolate Plasmid]
Spin  @
Centrifuge: 17900 34
?. [Isolate Plasmid]
Re-load and re-spin until everything is loaded.
?. [Isolate Plasmid]
Buffer PB to each column
500 µl
?. [Isolate Plasmid]
Spin  @
Centrifuge: 17900 34
?. [Isolate Plasmid]
750 uL Buffer PE
?. [Isolate Plasmid]
Spin  @
Centrifuge: 17900 34
?. [Isolate Plasmid]
Spin again to dry  @
Centrifuge: 17900 34
?. [Isolate Plasmid]
Elute with 30-50 uL H2O
?. [Isolate Plasmid]
(Optional) Re-load H2O on columns and re-spin   @  to increase yield.
Centrifuge: 17900 34
?. [Grow bacteria containing plasmid]
Pick colony from plate. Inoculate 50 mL LB broth per vector.
TITLE: Make liquid Superbroth for bacteria
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare Superbroth.</div><div class = "text-block">Superbroth is a liquid medium to feed bacteria.</div></div>

[STEPS]
?. 
?. ABCD1Ingredients  Quantity2Bacto-tryptone 72g3Yeast
  Extract144g4Glycerol24ml5Double
  distilled H2Oupto 5.4L
ABCD1Ingredients  Quantity2Bacto-tryptone 72g3Yeast
  Extract144g4Glycerol24ml5Double
  distilled H2Oupto 5.4L
?. A1Before starting ensure six 1L bottles have been
  sterilised.2Fill a 5.4L&nbsp;
  bell jar with 3 litres of double distilled H2O. and mark off. Then&nbsp;3Dissolve all the ingredients and then make up to
  the 5.4L mark with&nbsp; double distilled H2O.4Dispense 900ml into six sterile 1L bottles.
  Label,date and autoclave.
A1Before starting ensure six 1L bottles have been
  sterilised.2Fill a 5.4L&nbsp;
  bell jar with 3 litres of double distilled H2O. and mark off. Then&nbsp;3Dissolve all the ingredients and then make up to
  the 5.4L mark with&nbsp; double distilled H2O.4Dispense 900ml into six sterile 1L bottles.
  Label,date and autoclave.
TITLE: Preparation of nasopharyngeal samples for immunofluorescence assay
AUTHORS: Maria Lucia Silva, Flavia Escremim de Paula, Talita Gagliardi, Eurico Arruda

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Add the nasopharyngeal sample into a conical tube.
?. Only in samples with much mucus, drop N-L-Acetyl-Cysteine (maximum 1ml) into the tube and homogenize by vortex.
?. Centrifuge the tube for 10 minutes at 4oC  and  300 x g.
?. Discard the supernatant.
?. Homogenize the pellet of cells with phosphate-buffered saline solution 1x (PBS 1x).
?. Drop 5ul of diluted suspension of cells into a glass slide.
?. Let the slide inside the hood or in front of a hair dryer until the sample dries.
?. Immerge the glass slide into pure and cold acetone (fixing solution).
?. After 5 minutes, wash the slide one in PBS 1x.
?. Store the glass slides at -20oC.
TITLE: Unbiased Stereology (MBF Bioscience, StereoInvestigator Software)
AUTHORS: Jhodi Webster, Ashley Harms

[DESCRIPTION]
This protocol allows for unbiased stereo logical assessment of neurons and other cells in specific brain regions using StereoInvestigator Software. 

[STEPS]
SECTION: WORKFLOW
1. Create a map of your slide using the stereology worksheet and make note of the estimated population and total markers counted
SECTION: WORKFLOW
2. Set slide properly on the microscope. 
SECTION: WORKFLOW
3. Click on Probes > Optical Fractionator Workflow > Start a new subject
Label subject/study
Existing sample in configuration: no
Number of sections to count:__*see table
Enter cut thickness: 40μm
Section Evaluation Interval: __*see table
Start with number one
z-value of first section = 0
SECTION: WORKFLOW
4. In Select Low Mag Lens, click 5X > Next Step
SECTION: WORKFLOW
5. Trace the Contours of Interest for each section on your slide. If you need more contours for other brain regions, label them in preferences. 
SECTION: WORKFLOW
6. Select High Mag Lens select 40X (use light focuser on) > Next Step
SECTION: WORKFLOW
7. Select 'manually enter the average mounted thickness'. Average Mounted thickness is 25μm
SECTION: WORKFLOW
8. Define the counting frame (see the guidelines for more specs): your goal is to have 5-10 cells for each counting frame and about 20-25 sites/region
SECTION: WORKFLOW
9. Manually enter grid size (see the guidelines for more specs): Enter 15 percentage. Approximate sites = 1 > Display changes. 
SECTION: WORKFLOW
10. Optical Director Height > program will calculate this based on settings > Manual Focus > Next Step
SECTION: WORKFLOW
11. Count objects: Select your first section > Select the desired region > Start counting 
SECTION: WORKFLOW
12. Focus on the top of the tissue > OK (if you struggle with this, find the bottom of the section and then count up 20 units from there)
SECTION: WORKFLOW
13. Select your markers according to the desired cell types (these can also be customized)
For each site, scroll up and down throughout the z plane and adjust brightness
Count cells with visible nucleus in the square. If a cell touches a line of the counting frame: if it touches the green line, count the cell; if it touches a red line, do NOT count it. 
SECTION: WORKFLOW
14. Once finished with counting: Select "Completed Counting" To view results and export data to excel: View Results > Export to Excel 
SECTION: WORKFLOW
15. To go to the new reference icon in the top left corner and repeat steps.
TITLE: Enzymatic PCR Cleanup Protocol (NEB #M0525)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Rapid and irreversible heat inactivation eliminates unwanted activity</li><li style = "counter-reset:ol0;list-style-type:disc;">Improved storage stability versus native enzyme</li><li style = "counter-reset:ol0;list-style-type:disc;">Faster reaction setup (no supplemental additives like zinc required) and shorter incubation time</li><li style = "counter-reset:ol0;list-style-type:disc;">Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)</li><li style = "counter-reset:ol0;list-style-type:disc;">Less enzyme required (high specific activity), resulting in a lower cost per reaction</li><li style = "counter-reset:ol0;list-style-type:disc;">No need for multiple phosphatases (Quick CIP removes 5′- and 3′- phosphates from DNA, RNA and dNTPs )</li><li style = "counter-reset:ol0;list-style-type:disc;">Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis</li><li style = "counter-reset:ol0;list-style-type:disc;">Recombinant for purity, consistency and value</li></ul></div></div>

[STEPS]
?. Add  of Quick CIP (NEB #M0525) to  of PCR product.
1 µl
5 µl
Note that 0.5 μl of Exo I (NEB #M0293) and 1 μl of rSAP (NEB #M0371) can be used in the same volume.
?. Incubate the mix at the  for .
37 °C
?. Inactivate both enzymes at  for .
80 °C
?. PCR products are ready for downstream application.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The thermocoagulation model is a type of focal cerebral ischemia with craniectomy. It represents an opportunity to study permanent ischemic brain lesions with good reproducibility and low mortality</p>

[STEPS]
?. 
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?.
TITLE: General procedure for long-term cryopreservation  of Yeast
AUTHORS: Andreas Sagen

[DESCRIPTION]
A simple and generalized procedure for freezing yeast cells.

[STEPS]
SECTION: 50% Glycerol solution
1. Fill a 200 mL Duran bottle with 100 mL PBS

Materials:
 
SECTION: 50% Glycerol solution
2. Transfer 100 mL 100% Glycerol to Duran bottle

Materials:
 
SECTION: 50% Glycerol solution
3. Sterilize by autoclave at 121 °C for 15 min
4. Incubate a 20 mL batch of Yeast overnight

 

5. Aliquot saturated culture into an appropriate container. For example:
    60 µL saturated culture in a 200 µL reaction tube
    600 µL saturated culture in a 1.5 mL reaction tube
    900 µL saturated culture in a 2.0 mL cryotube
6. Mix in 50% glycerol solution. For example:
    40 µL saturated culture in a 200 µL reaction tube
    400 µL saturated culture in a 1.5 mL reaction tube
    600 µL saturated culture in a 2.0 mL cryotube
7. Transfer to an ultra-low temperature or vapor-phase nitrogen freezer for long-term storage
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>Glassware should be acid-washed and rinsed well with dH<sub>2</sub>O before use. If unable to measure small quantities accurately, either scale up the recipe or prepare stock solutions of the individual compounds.</p>

[STEPS]
?. 
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?.
TITLE: Stedehouder, J. and Roberts, B.M. et al. (2024) Rapid modulation of striatal cholinergic interneurons and dopamine release by satellite astrocytes 
AUTHORS: Jeff Stedehouder, Bradley M. Roberts, Shinil Raina, Alan K. L. Liu, Laura Parkkinen, Dorly Verdier, Arlette Kolta, Stephanie J Cragg

[DESCRIPTION]
This collection contains seven protocols detailing methods used in Stedehouder, J. and Roberts, B.M. et al (2024) Rapid modulation of striatal cholinergic interneurons and dopamine release by satellite astrocytes.

[STEPS]
TITLE: Transformation of Diplonema papillatum using V5+NeoR construct
AUTHORS: Binnypreet Kaur, Drahomíra Faktorová, Julius Lukes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>p57-V5+Neo</span><span style = "vertical-align:super;">R </span><span>plasmid contains V5-tagged aminoglycoside 3'-phosphotransferase gene (APT; conferring resistance to neomycin) flanked by partial regulatory sequences derived from the hexokinase gene of the kinetoplastid </span><span style = "font-style:italic;">Blastocrithidia</span><span> (strain p57).</span></div><div class = "text-block">Plasmid GenBank accession number = MN047315</div></div>

[STEPS]
?. p57-V5+NeoR plasmid contains V5-tagged aminoglycoside 3'-phosphotransferase gene (APT; conferring resistance to neomycin) flanked by partial regulatory sequences derived from the hexokinase gene of the kinetoplastid Blastocrithidia (strain p57).
?. This 1779 bp long sequence is surrounded by SwaI restriction sides and was cloned in the pBluescript II SK(+) backbone.
?. Before transformation, the p57-V5+ NeoR plasmid was cut using SwaI, run on the gel and a fragment of 1779 bp containing p57-V5+ NeoR construct was gel purified
?. 3 ug of purified construct was electroporated into D. papillatum cells using an established protocol (Kaur et al,. 2018). About 18 h after electroporation, a scale of 45 – 83 ug/ml G418 (Geneticin) was added to the media and after about 2 weeks G418-resistant clones were recovered.
TITLE: Purification of FUNDC1-GST
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of FUNDC1-GST.

[STEPS]
SECTION: Purification - FUNDC1-GST
1. To purify FUNDC1-GST, fuse the cytosol-exposed domain of FUNDC1 (1-50aa) to a C-terminal GST-tag through cloning into a pET-DUET1 vector (available on Addgene). 
SECTION: Purification - FUNDC1-GST
2. Introduce the point mutants by in vitro mutagenesis to generate FUNDC1 Y18A/L21A (ΔLIR)(available on Addgene). After the transformation of the pET-DUET1 vector encoding FUNDC1-GST wild-type or mutants in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600  of 0.4 and then continue at 18 °C. 
SECTION: Purification - FUNDC1-GST
3. Once the cells reaches an OD600 of 0.8, induce protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 

SECTION: Purification - FUNDC1-GST
4. Collect the cells by centrifugation and resuspend in lysis buffer. 

Lysis buffer:

 
 
SECTION: Purification - FUNDC1-GST
5. Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification - FUNDC1-GST
5.1. Sonicate the cell lysates for 30 s (1/2).
SECTION: Purification - FUNDC1-GST
5.2. Sonicate the cell lysates for 30 s (2/2).
SECTION: Purification - FUNDC1-GST
6. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °Cwith gentle shaking to bind FUNDC1-GST. 
SECTION: Purification - FUNDC1-GST
7. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - FUNDC1-GST
8. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash buffer:
 
 
 
High salt buffer:
 
 
 
SECTION: Purification - FUNDC1-GST
9. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolves in wash buffer at 4 °C, to elute FUNDC1-GST from the beads. 
SECTION: Purification - FUNDC1-GST
10. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - FUNDC1-GST
11. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - FUNDC1-GST
12. Pool the supernatant fractions, filtered through a 0.45 µm syringe filter, concentrated with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 
SECTION: Purification - FUNDC1-GST
13. Elute the proteins with SEC buffer. 

 SEC buffer: 

 
 
SECTION: Purification - FUNDC1-GST
14. Analyze fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified FUNDC1-GST. 
SECTION: Purification - FUNDC1-GST
15. After concentrating the purified protein, aliquote the  protein and snap-frozen in liquid nitrogen.
TITLE: Homology modeling with trRosetta
AUTHORS: Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for homology modeling using trRosetta written for students in Biochemistry I at James Madison University. </div></div>

[STEPS]
?. [Setting up modeling]
Navigate to the trRosetta homepage.
?. [Setting up modeling]
Input your sequence in FASTA format. The first list should start with > as shown in the example below.
?. [Setting up modeling]
Enter your email address and give a target name.
It is always a good idea to include target name or identifier in case you have multiple sequences that you are predicting.
?. [Setting up modeling]
Adjust any options that you want and record these changes as a note on this step.
?. [Setting up modeling]
Press submit and wait for the job finish email. Typically takes 2 to 24 hours to get a result.
?. [Modeling results]
When your receive the job finished email, navigate to the results link.
Help guide for trRosetta: https://yanglab.nankai.edu.cn/trRosetta/help/
?. [Modeling results]
The top model will be shown in a window like below with an estimated TM score.
The TM-score is a template model score and measures the similarity between two proteins. 0 is no match, while 1 is a good match. Models with scores >0.5 are likely to be reliable.
?. [Modeling results]
The summary indicates how the model was built during the modeling along with links to the data that was used during modeling and the 4 "next best" models.
?. [Modeling results]
The predicted 2D information shows the predicted long range interactions within the structure along with information on the dihedral angles.
This information is good for quickly identifying interesting inter-domain interactions or potential sites of regulation.
?. [Modeling results]
The predicted 1-D information shows the secondary structure prediction and predicted regions of disorder.
?. [Saving data]
At the top of the window you can download the entire file in .tar.bz2 format. To open this file it must be decompressed.Alternatively, you can download the relevant models and data individually from the summary table.Save files as [date]_[proteinname]_[teamname]_trRosettaReplace [proteiname] with the target protein name, [teamname] with your name or your team's name, [date] with the date.Ex.: 20210810_UFL1_Berndsen_trRosetta
?. [Saving data]
Indicate WHERE you saved the file as a note on this step.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol consists of an IgG purification step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously purified, proceed directly to the cross-linking protocol.

[GUIDELINES]
<strong>Overview</strong><br /><br /><strong>Materials Needed:&nbsp;</strong><br /><br />Protein A (<a href='https://www.neb.com/products/s1425-protein-a-magnetic-beads'>NEB #S1425S</a>&nbsp;) or Protein G (<a href='https://www.neb.com/products/s1430-protein-g-magnetic-beads'>NEB #S1430S</a>&nbsp;) Magnetic Beads&nbsp;<br />Elution Buffer: 0.1 M glycine-HCl (pH 2.5)&nbsp;<br />Binding Buffer: 0.1 M NaPhosphate Buffer (pH 8.0)<br />Dimethyl pimelidate dihydrochloride (Sigma, D-8388) dissolved at 25 mM in Cross-linking Buffer.&nbsp;<br />Cross-linking Buffer: 0.2 M triethanolamine (pH 8.2)&nbsp;<br />Blocking Buffer: 0.1 M ethanolamine (pH 8.2)&nbsp;<br />Immunoglobulin in Binding Buffer&nbsp;<br /><br />This protocol consists of an IgG purification step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously purified, proceed directly to the cross-linking protocol.

[STEPS]
?. 
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TITLE: 16s rDNA sequencing reaction and precipitation protocol 
AUTHORS: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones

[STEPS]
?. [Sequencing reaction ]
Consider a total volume of 10 µl
?. [Sequencing reaction ]
Add 6 µl of water for PCR
?. [Sequencing reaction ]
Add 2 µl of save money buffer
?. [Sequencing reaction ]
Add 1 µl of DNA (10 - 20 ng/µl)
?. [Sequencing reaction ]
Add 0.5 µl of primer
?. [Sequencing reaction ]
Add 0.5 µl of Byg Dye
?. [Sequencing reaction ]
Place the reaction in the thermal cycler with the following cycling program:
?. [Precipitation]
Add 1 µl of EDTA (125 mM) to each well
?. [Precipitation]
Add 1 µl of 3M Sodium Acetate (NaOAc) to each well
?. [Precipitation]
Add 25 µl of 100% cold Ethanol to each well, cap tightly and vortex slightly
?. [Precipitation]
Incubate at 4 °C for 15 minutes
?. [Precipitation]
Centrifuge at 2250g for 30 minutes
?. [Precipitation]
Dispense the contents in the sink immediately and invert immediately on paper towels
?. [Precipitation]
If it is not dispensed immediately, centrifuge at 2250g for 2 min
?. [Precipitation]
Additional 35 µl of 70% chilled ethanol in each well
?. [Precipitation]
Centrifuge at 2250g for 15 minutes
?. [Precipitation]
Dispense the contents in the sink and centrifuge the inverted plate 15g for 1 minute
?. [Precipitation]
Incubate the uncapped plate in the thermal cycler at 95 °C for 5 minutes
?. [Precipitation]
Resuspend the samples in 10 µl of Hi-Di formamide, cover with the cap and vortex slightly
?. [Precipitation]
Incubate in the thermal cycler at 95 °C for 2 minutes
?. [Precipitation]
Put ice for 2 minutes
?. [Precipitation]
Place the card in the sequencer
?. [Sequencing reaction ]
Run an Initial denaturation step of 1 min at 96 °C.
?. [Sequencing reaction ]
Run 30 cycles of 15 sec at 96 °C for denaturation, 15 sec at 50 °C for annealing, 4 min at 60 °C for extension.
?. [Sequencing reaction ]
Store amplification products at 4°C until further use in precipitation reactions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
These are protocols from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[STEPS]
?.
TITLE: Salivary collection participant instructions using OMNIgene ORAL OM 501 device
AUTHORS: Elisa Menozzi, Roxana Mezabrovschi, Sara Lucas, Jane Macnaughtan, Sofia Koletsi, Aleesa Nazeer, Nadine Loefflad

[DESCRIPTION]
This is an adapted protocol for collection of salivary samples from participants. This protocol was adapted from DNA Genotek's instructions available at: https://www.dnagenotek.com/us/support/collection-instructions/omnigene-oral/OM-501.html

[STEPS]
1.