eamag/protocols-with-steps huggingface dataset:
TITLE: Streaking and Isolating Bacteria on a LB Agar Plate
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Follow this protocol if you have a glycerol stock of the bacteria of interest and want to isolate an individual clonal population (single colony) of bacteria from this stock. Using a single colony from a freshly streaked agar plate to inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture of plasmids in your purified DNA. This protocol explains how to isolate a single bacterial colony by streaking it onto a LB agar plate.</div><div class = "text-block">This is a general protocol for streaking bacteria on LB plate and the following parameters need to be mentioned of a specific type of bacterial streaking:</div><div class = "text-block">1. Name of the bacterial strain</div><div class = "text-block">2. Growth temperature</div><div class = "text-block">3. Incubation time </div><div class = "text-block">4. Antibiotic resistance (if any)</div><div class = "text-block">5. Rpm of the shaking incubator</div></div>

[STEPS]
?. Obtain an LB agar plate with appropriate antibiotic if any.
?. Label the bottom of the plate with the strain name and the date. It is also a good idea to add the antibiotic resistance (if any) and your initials.
?. Sterilize your lab bench by spraying it down with 70% ethanol and wiping it down with a paper towel. Maintain sterility by working near a flame or bunsen burner.
?. Find the bacteria of interest from the bacterial stock database and obtain the appropriate glycerol stock from the -80C freezer (Freezer 6- Bacterial Stock box)
?. Using a sterile pipette tip touch the top of the glycerol stock to obtain the bacteria on the tip
?. Gently spread the bacteria over a section of the plate, as shown in the diagram below, to create streak #1Using a fresh, sterile pipette tip, drag through streak #1 and spread the bacteria over a second section of the plate, to create streak #2Using a third sterile pipette tip, drag through streak #2 and spread the bacteria over the last section of the plate, to create streak #3The repeated streaking is done to dilute the bacteria and obtain single colonies.
?. Incubate the plate (lid side down) with the newly plated bacteria overnight (generally 12-18 hours) at the appropriate growth temperature for the particular strain. In case of antibiotic resistant plasmids, incubation should not exceed the recommended duration as it poses risk of the bacteria metabolising the antibiotic.
?. Following adequate incubation, single colonies should be visible. A single colony should look like a white dot growing on the solid medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium. If the bacterial growth is too dense and you do not see single colonies, re-streak onto a new agar plate to obtain single colonies.
?. Store the streaked plates at 4C (lid side down to avoid condensation falling on the single colonies). The streaked plates can be used for up to a month to make liquid cultures.
TITLE: TruKeto Reviews Scam Exposed Honest Customer Results?
AUTHORS: wepixo  , H  H, health  , H  A

[DESCRIPTION]
https://www.eastbaytimes.com/2022/05/12/truketo-reviews-shark-tank-warning-tru-keto-pills-price-legit-website-for-sale/

[STEPS]
1. TruKetoLosing weight and overseeing down your extra muscle to fats sum has by no means, been expressly fundamental. In any case, with such a huge load of weight decline structures drifting around, it'll common be direct one which interminably works helpful. Which is the explanation such vast people are shuddering to get their fingers at the TruKeto Tablets. These keto tablets rely upon to organizations nearby the headway keto eating routine to help you with improving, swifter weight decline results. Furthermore, it could help you with doing it fundamentally more essential clear. These tablets should help you with moving into ketosis and eating your extra fats as a power source. In like manner, persevering through you're facilitated to view as extra, stay aware of to look at our TruKeto Review! Regardless, click on the standard beneathneath to peer what definite gives or starters are reachable for this phenomenal tablet sooner than guides of development are no more! TruKeto Weight Loss is the most outrageous current (amazing?) hankering for fats ingesting as much as hit the market. This tablet should help you with getting more important match staggeringly swifter and more imperative clean than a hint of the techniques you've been endeavoring. Besides, at the off hazard which you are appearing at an opening to defeat and have to vanish weight decline, the TruKeto Diet Pills might be in wellknown what you truly need to push through and start obtain results when more conspicuous. In any case, those tablets endeavor and advance out brief. Make a variable to apparatus the common beneathneath to get the total moving and spot what five star gives you could find while resources last. Click Here https://www.eastbaytimes.com/2022/05/12/truketo-reviews-shark-tank-warning-tru-keto-pills-price-legit-website-for-sale/
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a generic&nbsp;PNGase F protocol with denaturing reaction conditions. It is appropriate for both&nbsp;<a href="https://www.neb.com/products/p0704-pngase-f" target="_blank">P0704</a>&nbsp;and&nbsp;<a href="https://www.neb.com/products/p0708-pngase-f-recombinant" target="_blank">P0708</a>.

[BEFORE_START]
Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are below.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to calculate the hyperemic myocardial blood flow, a clinically critical parameter, with CZT-based SPECT and a single-scan rest/stress protocol that is proposed in our manuscript.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This collection of protocols describes the preparation of reagents for transfecting DNA into the choanoflagellate <em>Salpingoeca rosetta</em> using a Nucleofector 4d Device and SF kit from Lonza. Plasmids that use <em>S. rosetta</em> promoters and regulatory elements to drive the expression of luciferase and fluorescent protein reporters are being deposited at Addgene. </p>
<p> </p>
<p>Acknowlegements: Arielle Woznica and Laura Wetzel for meticulous editing of protocol and clarifying steps in the procedure. The Gordon and Betty Moore Foundation for supporting this research.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Manufacturer's protocol</p>

[STEPS]
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TITLE: Direct acid extraction
AUTHORS: Sigrid Verhelst, Laura Corveleyn

[DESCRIPTION]
Protocol to extract histones from cells using direct acid extraction with the purpose of label-free LC-MS/MS measurement.

[STEPS]
1. Resuspend the pellet in 0,4 N HCl by soft pipetting until no clumps are left in solution (125 µL for 1x106cells)
 

2. Incubate 4h in acid on rotator 4°C to promote lysis of nuclei and solubilization of histones. 
 
3. Spin for 10 minutes at  4°C, 16000 g. 
4. Transfer supernatant to new Eppendorfs.
 

5. Add, drop by drop (very slowly and in the middle of the eppendorf), TCA (final concentration 33%: 61.25µl for 1x106) to promote precipitation of histones and invert the tube several times.
6. Incubate on ice for 30 minutes. 
7. Spin for 10 min at 4°C, 16000g to pellet the histones.
 

8. Remove the supernatant.
 

9. Add ±150 µL ice-cold acetone (don’t resuspend the pellet) to remove TCA. 
10. Spin for 5 min at 4°C, 16000g. 
11. Remove the supernatant.
12. Add ±150 µL cold acetone (don’t resuspend the pellet) to remove TCA. If not all the acid is removed it will destroy the SDS-gel. 
13. Spin for 5 min at 4°C, 16000g. 
14. Remove the supernatant.
15. Dry at room temperature for 30 minutes (until no acetone left). 
16. Resuspend in milliQ water (50 µL for 1x106 cells).
17. Transfer 20 µL (from 400.000 cells) to a new Eppendorf tube for gel. 
18. Vacuum dry samples.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<br />(store at 4 degrees Celsius)<br />80% Ethanol<br />4% Formaldehyde<br />5% Acetic Acid<br />1% Glutaraldehyde<br /><br />Measured Instructions<br />Total volume 250 ml FAAG:<br />200 ml 100% Ethanol<br />27.5 ml 37% Formaldehyde<br />12.5 ml Glacial Acetic Acid<br />10 ml 25% Glutaraldehyde (EM Grade)

[STEPS]
TITLE: Expression and purification GST-tagged ATG13:ATG101 constructs
AUTHORS: Adam      Yokom, Xuefeng  Ren

[DESCRIPTION]
Expression and purification of GST-tagged ATG13/ATG101 constructs

[STEPS]
SECTION: Expression
1. Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
   
SECTION: Purification
8. Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA) 
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Purification
9. Clarify lysate for 17000 rpm, 35 min, 4 °C

SECTION: Purification
12. Rock supernatant with equilibrated resin for 60 min at 4 °C
SECTION: Purification
11. Load supernatant onto resin using a gravity column setup
SECTION: Purification
13. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
SECTION: Purification
15. Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA)
SECTION: Purification
14. Elute with lysis buffer plus 25 mM glutathione for GST resin
    
SECTION: Purification
16. Pool peak fractions, concentrate, snap freeze, and store at -80C 
SECTION: Expression
3. In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
4. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C 

SECTION: Expression
5. Add mixture to cells. Let cells grow for 2880 min 

SECTION: Expression
6. Harvest Cells 500 rpm, 10 min, 4 °C 

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at -80C until purification or lyse immediately 
SECTION: Purification
10. Wash GST Sepharose 4B resin into lysis buffer (without Triton)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>This protocol was designed and developed at this laboratory.</p>
<p>The assay specifically targets the 3' UTR region of DENV-1 and is designed as a qualitative screening test for human cases of DENV-1 infection, but not other known DENVs.</p>
</div>

[BEFORE_START]
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familar with the thermocycler and software used to run the protocol and with PCR in general..</li>
</ul>
</div>
</div>

[STEPS]
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TITLE: Targeted Expansion Sequencing Protocols
AUTHORS: Anubhav    Sinha, Yi  Cui, Shahar        Alon, Fei  Chen, Asmamaw      T. Wassie, Ed  Boyden

[DESCRIPTION]
This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry.

For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

[STEPS]
TITLE: Purification of GST-FAM134C
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of GST-FAM134C and its analysis.

[STEPS]
SECTION: Purification procedure
1. To purify GST-FAM134C, fuse the cytosol-exposed domain of FAM134C (250-466aa) to a N-terminal synthesize the gene GST-tag by Genscript and clone into a pGEX-4T1 vector. Plasmid is also available from Addgene. 

SECTION: Purification procedure
2. After the transformation of the pGEX-4T1 vector encoding GST-FAM134C in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow 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 procedure
11. Elute the proteins with SEC buffer.

SEC Buffer:
 
 
 
SECTION: Purification procedure
10. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
9. To collect the supernatant, collect the beads by centrifugation. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 

SECTION: Purification procedure
8. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute GST-FAM134C from the beads. 

Wash Buffer:
 
 
 
SECTION: Purification procedure
7. Centrifuge samples to pellet the beads and remove the unbound lysate. Then wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash buffer:
 
 
 
Salt wash Buffer:
 
 
 
SECTION: Purification procedure
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 procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer. 

 Lysis Buffer:

 
 
SECTION: Purification procedure
3. Once the cells reach an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 

SECTION: Purification procedure
6. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-FAM134C. 

SECTION: Purification procedure
12. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified GST-FAM134C. 

SECTION: Purification procedure
13. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
14. Store proteins at -80 °C.
TITLE: Liposome preparation
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the preparation of 100% PS liposome and GC/PS lipid nanotubes used for LRRK2 binding, tubulation assays.

[STEPS]
SECTION: Liposome preparation
1. Dissolve lipid mixtures with chloroform in glass vials in moles percent as follows:

PS liposomes: 99.5% brain PS:0.5% Rhod-PE.
GC/PS nanotubes: 39.5% Galactosylceramide:60% brian PS:0.5% Cy5-PE.
SECTION: Liposome preparation
2. Evaporate chloroform under a stream of nitrogen gas to produce a lipid film on the glass surface.
SECTION: Liposome preparation
3. Dry the lipid film in a vacuum oven for 60 min.
SECTION: Liposome preparation
4. Rehydrate the dried lipid films in liposome buffer at a final concentration of 1 mg/mL (~1.2 millimolar (mM)).
SECTION: Liposome preparation
5. For PS mixtures, form the liposomes by three freeze (liquid N2)–thaw (37 °C water bath) cycles.
SECTION: Liposome preparation
6. Remove large aggregates by a brief centrifugation (500 x g for 5 min) and store in the dark at 4 °C to avoid photooxidation.
SECTION: Liposome preparation
5.1. For GC/PS mixtures, form the lipid nanotubes by a brief vortexing instead of freeze-thaw cycles.
TITLE: Transformation of competent E. coli
AUTHORS: yasoo 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Marburg iGEM 2021 Team's Competent E. coli transformation protocol</div></div>

[STEPS]
?. Briefly thaw chemically competent E. coli cells on ice.
?. Add ∼  of plasmid-DNA to the cells. Alternatively just use  for retransformations and  for freshly cloned constructs (Golden Gate reactions).
100 ng
[or 2 µl]
5 µl
?. Incubate on ice for  (can be skipped for retransformations).
?. Heat shock the cells at  for  (30 seconds for NEB® Turbo cells).
42 °C
?. Recover on ice for , then add  of  to the cells. Alternatively use LB medium, but SOC is better for the cells.
500 µl
?. Incubate at  for 1 hour (if construct confers amp resistance) or 2 hours (if construct confers kan, cam, tet or spec resistance). Use the incubation time to prepare the plates and pre-warm them in an incubator.
37 °C
?. Plate on pre-warmed agar plates containing the corresponding antibiotic.
100 µl
?. Optionally you can centrifuge for at , resuspend the pellet in the remaining ∼ and plate it.
Centrifuge: 3500 34
100 µl
TITLE: Tissue Sectioning Guidelines (FFPE) - CODEX/PhenoCycler
AUTHORS: Santhosh Sivajothi

[DESCRIPTION]
The purpose of this SOP is to provide general guidelines for preparing FFPE tissue sections suitable for PhenoCycler/CODEX

[STEPS]
SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
1. Prepare the microtome for sectioning. Section the tissue at a thickness of 5 μm.

 

SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
2. For best results, the tissue should be completely adhered to the slide with minimal tears or folds.
SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
3. Gently place the tissue section in the center of the slide within the imageable area as shown in
grey rectangle below. 
If tissue overlaps the boundary, proper coverslip seal will not be formed. Multiple tissues can be placed within the boundary, as long as there is no overlap of paraffin over adjacent tissues

  

SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
4. Example of good tissue placement: 

            

SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
5. Dry tissue sections overnight at RT
SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
6. After drying, store cut slides at 4°C (for up to six months)
SECTION: Tissue Sectioning Guidelines for CODEX/PhenoCycler
7. NOTE: Do not place any stickers or labels on the frosted end of the slide as it may interfere with coverslip adhesion. Use markers or directly print labels on the slide.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This procedure is optimized for the isolation of 10<sup>6</sup> cells per test. If working with fewer than 10<sup>6</sup><br />cells, keep volumes as indicated for 10<sup>6</sup> cells. For best results, optimize the conditions to your specific cell number and<br />tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p><strong>Product description and procedure summary:</strong><br />Target cells are positively selected or depleted by incubating the sample with the biotin anti‐mouse CX3CR1<br />antibody followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is<br />retained by the use of a magnetic separator. These are the CX3CR1+ cells, do not discard them if those are the<br />cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic<br />characterization, etc.<br /><br /><br /></p>

[GUIDELINES]
<p><strong>Reagents and instruments required:</strong><br />MojoSort™ Buffer (5X) (Cat. No. 480017)<br />MojoSort™ Magnet (Cat. No. 480019/480020) or compatible magnetic separation system<br />Adjustable pipettes<br />70 μm filters (one per sample)<br />5 mL (12 x 75 mm) or 14 mL (17 x 100 mm) polystyrene tubes<br />Reagents for sample preparation<br />Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/faqecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/faqecje.jpg" data-ofn="mojosort mouse cx3cr1.JPG" /></p>

[STEPS]
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TITLE: GNRI's COVID-19 Antigen Lateral Flow Test Device Use Steps
AUTHORS: Dr Georgios Gerardos

[STEPS]
?. [Please follow all steps]
Obtain a clean container (glass or cup) and produce a saliva sample in the container.
?. [Please follow all steps]
Remove a new COVID-19 Antigen Lateral Flow Test Device from its foil packaging.
?. [Please follow all steps]
Place the test strip in the container and leave for 10min.
?. [Please follow all steps]
Remove the test strip from the container and read results.Results interpretation:One Line (Control Line) = COVID-19 NegativeTwo Lines = COVID-19 PositiveNo Lines = Invalid Test. Please repeat test.One Line (Test Line) = Invalid Test. Please repeat test.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>- Use with Ultra Streptavidin Detection Kit (SIG-32250) or (SIG-32248)</p>
<p> </p>
<p>- Positive control: Normal human skin</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>CZV8-top agar</strong> </p>
<p>                                            <strong><u>100 mL</u></strong> per transformation                                     <strong><u>200 mL</u></strong></p>
<p>Czapek-Dox liquid                4.54 g                                                                     9.08 g</p>
<p>agar                                      0.75 g                                                                     1.5 g</p>
<p>sorbitol                                 18.22 g                                                                   36.44 g</p>
<p>centrifuged V8 juice             20 mL                                                                     40 mL</p>
<p>add H<sub>2</sub>O to                          100 mL                                                                    200 mL</p>
<p> </p>
<p>adjust to pH 6.0 with NaOH           </p>
<p>split batch in 2  (½ for plating protoplasts, ½ for overlay)</p>
<p>autoclave</p>

[STEPS]
?.
TITLE: 16S Bacteria 338F-516P-805R BSA
AUTHORS: Roey Angel, Eva Petrova, Ana Lara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Universal 16S rRNA BActeria 338F-516P-805R</div><div class = "text-block"><span>BAC338F      ACT CCT ACG GGA GGC AG , target </span><span style = "font-style:italic;">E.coli :</span><span>  338-354, Yu et al. (2005), B&B</span></div><div class = "text-block"><span>BAC516P*    TGC CAG CAG CCG CGG TAA TA, target </span><span style = "font-style:italic;">E.coli :</span><span>  516-536, '</span></div><div class = "text-block"><span>BAC805R      GAC TAC CAG GGT ATC TAA TC , target </span><span style = "font-style:italic;">E.coli :</span><span>  785-805, '</span></div><div class = "text-block">* Probe must be dual-labelled either with 5’-6-FAM, 3’-BHQ1 or any other valid combination.</div></div>

[STEPS]
?. [QPCR mixture]
ABCD1ReagentFinal conc.1 tube (20μl)plate (20μl x 100)2PCR H2O&nbsp;4.64603iQTM Supermix1x1010004MgCl2 (25mM)4.0 mM0.8*80*5BSA (20μg/μl)0.2 μg/μl0.2206338F (10μM)0.5 μM1.01007805R (10μM)&nbsp;0.5 μM1.01008516P (10μM)†0.2 μM0.4409Template&nbsp;22 x 100* Buffer contains  MgCl2 at final conc. of 3.0 mM
ABCD1ReagentFinal conc.1 tube (20μl)plate (20μl x 100)2PCR H2O&nbsp;4.64603iQTM Supermix1x1010004MgCl2 (25mM)4.0 mM0.8*80*5BSA (20μg/μl)0.2 μg/μl0.2206338F (10μM)0.5 μM1.01007805R (10μM)&nbsp;0.5 μM1.01008516P (10μM)†0.2 μM0.4409Template&nbsp;22 x 100
?. [Program]
1.  95◦C – 5′2.  x 40 {       a.  95◦C – 30′′       b.  22◦C – 60′′  take a snapshot      }