eamag/protocols-with-steps huggingface dataset:
TITLE: Exp02
AUTHORS: Mayleth Medina Cárdenas, Estefanía Sauceda Camacho, Sofía Morga Benítez

[DESCRIPTION]
En este experimento se buscó analizar el cambio de la temperatura del agua con respecto a la ley cero de la termodinámica, de acuerdo al equilibrio térmico. Se planteó la posibilidad de una variación en el cambio de temperatura por el color del recipiente. Para corroborar dicha teoría, se realizó un experimento donde se colocó agua a tres termos exactamente iguales, pero de distinto color (negro, blanco y plateado) y con ayuda de un arduino uno y sensores de temperatura, se registraron los cambios de temperatura con respecto al tiempo de cada uno de los termos. También, se generó un modelo matemático a prueba y error que concordara lo mejor posible con las mediciones obtenidas. Se generó una gráfica que muestra la temperatura de cada uno de los termos con respecto al tiempo y este modelo para una comparación de estos. Finalmente, puede observarse un cambio en temperatura más lento dentro del termo blanco mientras que las del negro y el plateado son más similares. Asimismo, se calculó la potencia de cada uno de estos donde podemos apreciar que la del termo negro es menor que las otras dos.

[GUIDELINES]
Para su realización, se debe mantener una temperatura ambiente constante en todo momento. Además, asegurarse de las conexiones de los sensores y su correcto funcionamiento previo a la experimentación.

[STEPS]
SECTION: Circuito Arduino y software
1. Armar el siguiente circuito:

 

SECTION: Circuito Arduino y software
4. En el software de Arduino seleccionar la placa y puertos correspondientes.
SECTION: Circuito Arduino y software
4.1. Subir / cargar los programas en Arduino.
SECTION: Circuito Arduino y software
5. Configurar CoolTerms para registrar la lectura de datos en un archivo .txt.
SECTION: Circuito Arduino y software
3. Conectar el cable USB 2.0 a la placa de Arduino y a la laptop.
SECTION: Experimentación
6. Hervir agua y con esta vaciar 50 mL a cada uno de los termos, con la ayuda de un embudo.
7. Colocar un sensor de temperatura dentro de cada termo y asegurarse que esté en el fondo de cada uno.
9. La duración del experimento será hasta que la temperatura del agua de los tres termos alcance el equilibrio térmico.
SECTION: Resultados de mediciones
10. Las mediciones registradas en el archivo .txt obtenidas mediante CoolTerm se pasarán a un archivo Excel,
para crear con ellas una tabla, y posteriormente depurarlas.
SECTION: Resultados de mediciones
11. Subir el excel de las mediciones a Matlab, y con ellas generar una gráfica del cambio de temperatura de cada uno de los termos con respecto al tiempo.
8. Con el termómetro infrarrojo medir la temperatura ambiente.
SECTION: Circuito Arduino y software
2. Siguiendo el mismo acomodo, agregar una resistencia, un sensor térmico, y hacer sus respectivas conexiones.
TITLE: DQ-BSA quantification
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes DQ-BSA quantification.

[STEPS]
SECTION: DQ-BSA quantification
1. Segment maximal projection images from z-stacks spanning complete cells by using the find maxima function.
SECTION: DQ-BSA quantification
2. Threshold the duplicated images by default algorithm.
SECTION: DQ-BSA quantification
3. Combine the segmented and thresholder images by AND function to create a mask.
SECTION: DQ-BSA quantification
4. Obtain the mean gray values by applying analyze particle function to the mask and redirecting this whole analysis to the original images.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
This protocol comes from a group of other protocols. This protocol is (2) of (4): <br />1. <a href="https://www.protocols.io/view/Large-Volume-Marine-Cyanophage-Phage-Protocols-c3iykd" target="_blank">'Large Volume Marine Cyanophage Phage Protocols'</a><br />2. <a href="https://www.protocols.io/view/DNA-Extraction-Protocol-c32yqd" target="_blank">'DNA Extraction Protocol'</a><br />3. <a href="https://www.protocols.io/view/DNA-Precipitation-Protocol-c34yqv" target="_blank">'DNA Precipitation Protocol'</a><br />4. <a href="https://www.protocols.io/view/Checking-DNA-Concentration-with-Agarose-Gel-c35yq5" target="_blank">'Checking DNA Concentration with Agarose Gel'<br /></a><br />Needed:<br />
<ul>
<li>CsCl</li>
<li>Proteinase K</li>
<li>SDS</li>
<li>Incubator</li>
<li>Phenol</li>
<li>Microfuge @ 6,000rpm</li>
<li>Wide-bore pipette</li>
<li>Tube</li>
<li>Chloroform</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is an example of a simple short read mapping analysis that could be used as part of a transcriptomics or RNA-seq workflow.  </p>
<p>Code is intended for use on an Ubuntu 16.04 LTS OS, but it may work on other Unix or Unix-like systems.</p>
<p> </p>
<p>This tutorial uses data from the following very nice paper from the Hatfull group:</p>
<p>Dedrick, Mavrich, Ng, and Hatfull, Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives. BMC Microbiology, 2017. https://doi.org/10.1186/s12866-017-1131-2</p>
<p> </p>
<p>The tools that are used include:</p>
<p><strong>SRA toolkit</strong>: https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/</p>
<p><strong>bowtie2</strong>: http://bowtie-bio.sourceforge.net/bowtie2/index.shtml</p>
<p><strong>samtools</strong>: http://samtools.sourceforge.net/</p>
<p><strong>BEDOPS</strong>: https://bedops.readthedocs.io/en/latest/content/reference/file-management/conversion/bam2bed.html</p>
<p><strong>BEDtools</strong>: https://bedtools.readthedocs.io/en/latest/</p>
<p> </p>
<p>Make sure that these tools are installed before starting the tutorial. On a Ubuntu OS you should be able to install most with "sudo apt install", but you may wish to use a package manager such as Anaconda or Miniconda as well. </p>
<p>Throughout the tutorial some notes may be made about particular versions of tools. For example, note that the version of SRA toolkit that is installed with apt install often leads to downstream errors, it may be desirable to install this tool with Miniconda instead. </p>
<p> </p>
<p>Miniconda bash installers can be found here: https://conda.io/miniconda.html</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Preparation of staphylococcal protein-A conjugated to horseradish peroxidase.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This reagent can be used in ELISA, Western blotting and Dot blot to detect antigens and antibodies. It is important in the immunodiagnosis of infectious diseases. I find this useful in the detection of anti-HIV antibodies by ELISA.</div></div>

[STEPS]
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500 µg of staphylococcal protein-A (SpA) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
?. The mixture is incubated for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
?. The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
?. Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The conjugate is then stored at -20°C until further used.
TITLE: Whole Organoids Harvesting Procedure (Cultrex)
AUTHORS: Gabriela Vallejo Flores

[DESCRIPTION]
This protocol is use for whole organoids isolation, after isolation organoiods may be:
a. Resuspended in basement membrane matrix for further organoid culture.
b. Resuspended in freezing medium for cryopreservation.
c. Processed for biochemical analysis (such as RT-PCR, MS-PCR, sequencing, Western Blot, ELISA, or IHC)

[STEPS]
1. Working on ice, aspirate cell culture media and gently wash each well with 10 volumes of cold (2-8 °C) PBS (Table 1). Be careful not to disrupt the basement membrane matrix containing organoids.
2. Aspirate the PBS, and add 10 volumes of cold (2-8 °C) Cultrex™ Organoid Harvesting Solution to each well (Table 1).

 


3. Incubate the plate at 2-8 °C on a cold pack into a Polystyrene box for 60 minutes with moderate shaking. This incubation is complete when the basement membrane matrix dome is no longer visible at the bottom of the well and the organoids are seen floating at the bottom of the well. 
Note: Dislodging the dome with a cell scraper or pipet may accelerate this process.

4. Once the matrix depolymerizes, transfer the contents of the well into a tube on ice. Single wells may be transferred to a microtube while multiple domes may necessitate a 15 mL or 50 mL conical tube. Centrifuge the tube at 500 x g for 5 minutes at 2-8 °C in a swinging bucket rotor to pellet the organoids. Aspirate the supernatant.
5. Wash organoids with 10 volumes of cold (2-8 °C) PBS, and repeat centrifugation at 500 x g for 5 minutes at 2-8 °C in a  swinging bucket rotor to pellet the organoids. Aspirate the PBS.
6. Isolated organoids may be:
a. Resuspended in basement membrane matrix for further organoid culture.
b. Resuspended in freezing medium for cryopreservation.
c. Processed for biochemical analysis (such as RT-PCR, MS-PCR, sequencing, Western Blot, ELISA, or IHC)
TITLE: Shitty Models
AUTHORS: Thorsten Sommer, Nina Schiffeler, Sebastian Zachow, Laura Platte

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the description of how the Shitty Models approach can be integrated e.g. into a lecture. The corresponding article can be found at Github (</span><a href="https://github.com/SommerEngineering/blog-shitty-models" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/SommerEngineering/blog-shitty-models</span></a><span>) and on the website tsommer.education.</span></div></div>

[STEPS]
?. FramingIn this first step a context should be established. Why do we work with  Shitty Models now? What is the intention behind it? What are the  learning objectives? How is the method integrated into the course?
?. Kick-offAt this point the group can learn about the background, for example the  Shitty Robots. It also seems helpful to give a short talk on the topic  "Culture of Failure" and how failure can move us forward.
?. PreparationNext, the group can be familiarized with the available materials. If  there are rules for group work, they can also be applied in this step as  well. Obviously, it is important to ensure that the rules encourage  rather than hinder creativity, experimentation and exploration.
?. Shitty Model
Now the participants may build or program their Shitty Model. This step  can and should take up a large part of the available time.
?. PresentationAfter the models have been developed, the groups or individual  participants should present what they have achieved. This can be either  an entertaining show or a conventional presentation. The portfolio might  be presented, i.e. the different variants that may have been created.
?. FeedbackFinally, the learning objectives should be reflected upon so that the  participants become aware of their own learning process. There is no  kind of correction or direct assessment of Shitty Models: There is no  right or wrong characteristic for these models. This last step is also a  good place to start the exchange: What were best practices? What were  the lessons learned?
TITLE: MEMBER-OWNER 001
AUTHORS: rober  

[DESCRIPTION]
test

[STEPS]
1. test 

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Quisque hendrerit mauris quis nibh tincidunt pellentesque. Praesent congue justo vitae molestie dictum. Vivamus odio ipsum, consectetur eu nisi nec, posuere interdum metus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Nullam imperdiet dolor nec mi iaculis, sit amet molestie metus malesuada. Cras at nisi semper, varius nisi eu, maximus nunc. Proin mi justo, pulvinar nec ipsum nec, auctor faucibus ipsum.
Etiam fermentum magna non nibh eleifend, in cursus eros aliquet. Phasellus varius non odio vel sagittis. Fusce finibus diam id ex tempor, at pretium ipsum maximus. Aenean elementum placerat ligula vitae sodales. Quisque rutrum et elit et euismod. Quisque ut tellus nec orci blandit rutrum eu in tortor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Duis maximus, nibh ut auctor aliquam, enim libero tincidunt leo, non feugiat magna odio sed tortor.

2. test 2

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Quisque hendrerit mauris quis nibh tincidunt pellentesque. Praesent congue justo vitae molestie dictum. Vivamus odio ipsum, consectetur eu nisi nec, posuere interdum metus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Nullam imperdiet dolor nec mi iaculis, sit amet molestie metus malesuada. Cras at nisi semper, varius nisi eu, maximus nunc. Proin mi justo, pulvinar nec ipsum nec, auctor faucibus ipsum.
Etiam fermentum magna non nibh eleifend, in cursus eros aliquet. Phasellus varius non odio vel sagittis. Fusce finibus diam id ex tempor, at pretium ipsum maximus. Aenean elementum placerat ligula vitae sodales. Quisque rutrum et elit et euismod. Quisque ut tellus nec orci blandit rutrum eu in tortor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Duis maximus, nibh ut auctor aliquam, enim libero tincidunt leo, non feugiat magna odio sed tortor.

 

3. test 3

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Quisque hendrerit mauris quis nibh tincidunt pellentesque. Praesent congue justo vitae molestie dictum. Vivamus odio ipsum, consectetur eu nisi nec, posuere interdum metus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Nullam imperdiet dolor nec mi iaculis, sit amet molestie metus malesuada. Cras at nisi semper, varius nisi eu, maximus nunc. Proin mi justo, pulvinar nec ipsum nec, auctor faucibus ipsum.
Etiam fermentum magna non nibh eleifend, in cursus eros aliquet. Phasellus varius non odio vel sagittis. Fusce finibus diam id ex tempor, at pretium ipsum maximus. Aenean elementum placerat ligula vitae sodales. Quisque rutrum et elit et euismod. Quisque ut tellus nec orci blandit rutrum eu in tortor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Duis maximus, nibh ut auctor aliquam, enim libero tincidunt leo, non feugiat magna odio sed tortor.
TITLE: Q5® Site-Directed Mutagenesis (E0554)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554)</div></div>

[STEPS]
?. [Exponential Amplification (PCR)]
Assemble the following reagents in a thin-walled PCR tube.  ABC1 25 μl RXNFINAL CONC.2Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X310 μM Forward Primer1.25 μl0.5 μM410 μM Reverse Primer1.25 μl0.5 μM5Template DNA (1–25 ng/μl)1 μl1-25 ng6Nuclease-free water9.0 μl
ABC1 25 μl RXNFINAL CONC.2Q5 Hot Start High-Fidelity 2X Master Mix12.5 μl1X310 μM Forward Primer1.25 μl0.5 μM410 μM Reverse Primer1.25 μl0.5 μM5Template DNA (1–25 ng/μl)1 μl1-25 ng6Nuclease-free water9.0 μl
{"blocks":[{"key":"cv3on","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Exponential Amplification (PCR)]
Mix reagents completely.
?. [Exponential Amplification (PCR)]
Transfer to a thermocycler and perform the following cycling conditions:Thermocycling Conditions for a Routine PCR:   ABC1STEPTEMPTIME2Initial Denaturation98°C30 seconds325 Cycles98°C10 seconds450–72°C*10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C
ABC1STEPTEMPTIME2Initial Denaturation98°C30 seconds325 Cycles98°C10 seconds450–72°C*10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Assemble the following reagents:
{"blocks":[{"key":"9rkb4","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Mix well by pipetting up and down.
?. [Kinase, Ligase & DpnI (KLD) Treatment]
Incubate at room temperature for 5 minutes.
?. [Transformation]
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice.
?. [Transformation]
Add 5 μl of the KLD mix from the "KLD Section" above to the tube of thawed cells.
?. [Transformation]
Carefully flick the tube 4-5 times to mix. Do not vortex.
?. [Transformation]
Place the mixture on ice for 30 minutes.
?. [Transformation]
Heat shock at 42°C for 30 seconds.
?. [Transformation]
Place on ice for 5 minutes.
?. [Transformation]
Pipette 950 μl of room temperature SOC into the mixture.
950 µl
?. [Transformation]
Incubate at 37°C for 60 minutes with shaking (250 rpm).
?. [Transformation]
Mix the cells thoroughly by flicking the tube and inverting.
?. [Transformation]
Spread 50-100 μl onto a selection plate.
?. [Transformation]
Incubate overnight at 37°C
TITLE: DNA damage assessment in the adult Drosophila brain via pH2Av (S137) immunostaining
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes how to stain adult Drosophila brains with pH2Av to assess DNA damage.

[STEPS]
SECTION: Fly fixation and paraffin embedding
1. Fix flies of the desired genotype   in 4% formalin, embedded heads in paraffin, cut 
4-micron sections, and dry at 42˚C   

SECTION: Deparaffinization
2. Deparaffinize and bring through ethanol to water (xylene x 2, 100% ethanol x 2, 95% ethanol x 2, H2O x 2)
SECTION: Antigen-retrieval
3. Microwave slides in 10 mM sodium citrate for 15 min. Cool for 20 min. 
Stock: 100 mM sodium citrate, pH 6.0. Use at least  1 L of citrate solution in a large glass box to avoid drying

SECTION: Blocking
4. Block in blocking solution (PBS with 0.3% Triton and 2% dry milk) for 30min to 60 min
 
SECTION: Antibody incubation
5.  Incubate with primary antibody (Rockland pH2AV, cat # 600-401-914, dilution 1:100) overnight at room temperature
SECTION: Antibody incubation
6. Wash 3 x in PBST (PBS with 0.3% Triton)
SECTION: Antibody incubation
7. Incubate with fluorophore-coupled secondary antibody (Thermo Fisher) diluted 1:200 in blocking solution at room temperature

SECTION: Antibody incubation
8. Wash 2 x in PBST and 1 x in PBS
SECTION: Mounting
9. Mount in medium containing DAPI (Southern Biotech)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Potato tubers (250 g) were washed with distilled water and smashed in 500 mL of a mixture containing 50% alcohol and 1.6% citric acid. The resulting mixture was filtrated twice and centrifuged at 4000 rpm. The supernatant was transferred into a column filled with the pretreated macroporous resin AB-8 for 6 h, and the pigments absorbed in AB-8 were eluted with 95% alcohol. The eluate was concentrated with rotary evaporator at 30 °C and dried in vacuo for 12 h. Two biological replicates were used in this study.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Go  keto gummies Weight Loss: Reviews &amp; Is It Scam! Cost &amp; How To Consume!
AUTHORS: macallister.tevon  , macallister.tevon  

[DESCRIPTION]
Product Name - Go Keto Gummies
Composition –Natural Organic compound
Side-Effects–NA
Availability– Online
Rating –★★★★★
Official Website– Go Keto Gummies

This post in short, examines Go Keto Gummies, a well-known as well as popular keto gummies well-known intended for reduing obstinate body fat out of a great over weight entire body inside a secure and efficient way.
Go Keto Gummies are built to beat unwelcome body fat get in your system as well as assist most individuals obtaining preferred health and fitness as well as slimness. The item deals with more body fat build up in your system, as well as cleans away the item by any means costs. These include completely depending on ketogenic formulations that assist in cutting greasy compounds, carbohydrate food, fat laden calories, as well as adipocytes out of various areas of some sort of fat body.  

[STEPS]
TITLE: LB + Mg + Glucose for better Gram-N growth (agar + antibiotics possible) 1L
AUTHORS: Dylan brettingham

[DESCRIPTION]
LB media with additives to improve thegrowth of gram-negative microbes. LB is Mg defficeint and some of the proteins invovled in LPS synthesis require Mg as a co-factor, so by increasing the concentration the gram-N bacteria will be able to grow faster. LB also used to have glucose in the original formulation and this was removed, but the media is also pretty carbon starved and the glucose will simply aid in cell growth.

Blog Small Things Considered has a good post about this:
https://schaechter.asmblog.org/schaechter/2009/11/the-limitations-of-lb-medium.html

[GUIDELINES]
- Media out of the autoclave is very hot! Be careful
- The ingredients are fine powders for the most part and should not be inhaled

[STEPS]
SECTION: LB-2X media
1. Weigh out ingredients and add to clean bottle
- 10g Tryptone
- 5g Yeast Extract
- 5g NaCl (this is for Lennox formulation, 10g can be used for Miller Formulation)
- 2.46g MgSO4 * 7H20 (10mM)
- 10g D-glucose (1%)
+ 15g Agar if making plates (1.5%)



SECTION: LB-2X media
2. - Add stir bar to bottle and 1000mL of ddH20
- Stir until any chunks are dissolved
- Autoclave at temp (121C) for 40 minutes
    - If adjusting recipe to 500mL or below 30min autoclave is enough
SECTION: LB-2X media
3. Let cool (putting on a stir plate speeds this up) before pouring plates using aseptic technique
    - Can be poured in a laminar flow hood or biosafety cabinet
    - Heating the lip of the bottle well before pouring will help in preventing bubbles (they get popped as they pass over the hot lip)
    - Shaking the bottle will cause bubbles, try and not do this, use the stir plate
    - The thickness of the plates will affect how long they can be used for growth until they begin to dry, the thicker the longer they will last but will use more material
- If adding antibiotics, add them just prior to pouring not to the hot media out of the autoclave
    eg: if wanting 25ug/mL chloramphenicol in final media, add 1mL of 25mg/mL stock solution
SECTION: LB-2X media
4. Plates should be left out overnight and checked for growth the next day to be sure they are sterile
    - Labelling plates with media type before putting themaway will make life easier in the future!

Lightly spraying and wiping down outsdie of plates with 70% ethanol will help keep them sterile while being stored

Put plates away in the fridge (4C)
    - They will keep well for a month, and ive successfully used plates much older than that but its best to only make as much as you will use in a month
    - If antibiotics were added the 1 month limit is more strict as the antibiotic will loose efficacy over time
TITLE: Expression, purification and characterization of the GpC methyltransferase M.CviPI
AUTHORS: Karine Lapouge, Rozemarijn Kleinendorst, Kasit Chatsirisupachai, Nikolay Dobrev, Kim Remans, Arnaud   Krebs

[DESCRIPTION]
Methylation footprinting can be used to map protein-DNA contacts at the resolution of individual DNA molecules1. Enzymes with various nucleotide specificities have been successfully used to footprint genomes including GpC, CpG and A methyl-transfereases2–7. Among these M.CviPI methylate DNA in GpC context, that is distinct from CpGs that are endogenously methylated in mammals. This feature has been leveraged to profile nucleosome occupancy8,9; the binding of General Transcription Factors and RNA Pol II6; the co-occupancy of Transcription Factors (TFs)10 and the relation between TF binding and endogenous DNA methylation11. Here, we present a protocol for the production and purification of M.CviPI in E. coli. Our protocol routinely yields milligrams of protein at a quality and a concentration compatible with DNA footprinting applications. We characterize the purity and the activity of the purified enzyme, providing a benchmark for future production.

[STEPS]