eamag/protocols-with-steps huggingface dataset:
TITLE: WarmStart LAMP®
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to run nucleic acid amplification using the Warm-Start LAMP kit.</div><div class = "text-block">Each reaction produces 25 μL.</div><div class = "text-block"><span>For the original protocol, look at: </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;">.</span></div><div class = "text-block">*I have not verified the details of this protocol, I would like to remove it, but I cannot since it has been forked.</div><div class = "text-block">If you would like to use this protocol, I would recommend using the PDF above instead.</div></div>

[STEPS]
?. [Prepare Work Area]
Spray entire work area with 70% EtOH including pipettes, tip holder used for holding PCR tubes, and work surface. Wipe with a paper towel.
?. [Prepare Work Area]
Spray entire work area with RNAway.
?. [ Gather Materials]
Take styrofoam container to Marley 527 (directly across from Marley 509) and fill halfway with ice.
?. [ Gather Materials]
Set PCR tube holder on ice, and allow to cool for .
?. [ Gather Materials]
Transfer Master Mix, primers, RNAse free water, and target tubes from freezer to PCR tube holder on ice.
?. [ Gather Materials]
Allow reagents to thaw on ice
?. [ Gather Materials]
Carefully obtain (2) 0.2 mL PCR tubes. Label one with "NTC" and the other "TARG". These will be your reaction vessels.
To avoid contamination when grabbing PCR tubes, only touch the outside of tubes. Avoid touching the inside of the caps of other tubes in this process. This is critical.
?. [Prepare Reaction]
Using a 2-20 μL pipette, transfer RNAse free water into the TARGET reaction vessel.Using a 2-20 μL pipette, transfe RNAse free water into the NTC reaction vessel.
9 µl
10 µl
?. [Prepare Reaction]
Using a 0.5-10 μL pipette, transfer Primer to each reaction vessel.
2.5 µl
Primer will depend on what your target is. For E. Coli OH157, the primer tube is labelled with an 'F'.
?. [Prepare Reaction]
Using a 2-20 μL pipette, transfer WarmStart Master Mix into each reaction vessel.
12.5 µl
?. [ Gather Materials]
Vortex mix all reagents for approximately .
?. [Prepare Reaction]
Using a 0.5-10 μL pipette, transfer Target into the TARGET reaction vessel.
1 µl
?. [Prepare Reaction]
Vortex mix the reaction mixture.
?. [Prepare Reaction]
Spin down reaction mixture.
?. [Run LAMP Reaction]
Place reaction vessels into thermocycler.
?. [Run LAMP Reaction]
Turn on thermocycler
?. [Run LAMP Reaction]
Hit PROCEED to select a reaction cycle.
?. [Run LAMP Reaction]
Scroll using the '' keys to get to LAMP3.
?. [Run LAMP Reaction]
Press PROCEED to begin
?. [ Gather Materials]
Spin down all reagents for approximately .
TITLE: mxIF protocol
AUTHORS: Fan Wu

[DESCRIPTION]
This protocol is developed by SenNet JHU TMC group

[STEPS]
SECTION: Dewaxing and rehydration
1. Tissue slides are baking 1 hour before dewaxing
SECTION: Dewaxing and rehydration
3. Tissue slides are then dipped in water several times to finish rehydration
SECTION: Dewaxing and rehydration
2. Tissue dewaxing is performed with 5 minutes sequential wash in 3 times 100% xylene, 3
times 100% EtOH, followed by 90%, 80%, and 75% EtOH
SECTION: Heat antigen retrieval
4. Heat antigen retrieval steps are performed with 300 ul of unmasking solution in 32 ml of H2O in a steamer for 20 minutes
SECTION: Blocking
5. Blocking is performed with Peroxidase and Alkaline Phosphatase Blocking reagent for 15
minutes
SECTION: Primary antibody incubation
6. Primary antibody is incubated 1 hr at room temperature or 4 C overnight, depending on
targeted protein
SECTION: Signal amplification
7. Slides are incubated with 1 drop of anti-ms HQ (or anti-rb HQ depends on primary
antibody host animal) at room temperature for optimized time
SECTION: Signal amplification
8. Slides are incubated with 1 drop of anti-HQ HRP at room temperature for optimized time
SECTION: Signal amplification
9. TSA dyes are incubated for optimized time
SECTION: Antibody removal
10. Antibody removal kit or heat mediated antibody stripping is applied to remove the primary antibody
SECTION: Multiplex IF
11. Repeat step 5-10 until all the fluorescent channels are occupied
SECTION: Multiplex IF
12. Counter stain the slides and imaging with fluorescent microscope
SECTION: Bleaching
13. Bleaching is performed with 4.5% of H2O2 and 24mM NaOH made up with PBS (CycIF bleaching protocol)
14. Repeat step 5-13 for different rounds of imaging
TITLE: Histone extraction HLB protocol
AUTHORS: Sigrid Verhelst

[DESCRIPTION]
Protocol to extract histone proteins using a hypotonic lysis buffer for isolation of nuclei prior to acid extraction of histones that is easily amenable to label-free MS owing to the lack of detergents in the lysis buffer.

[STEPS]
SECTION: Isolate nuclei
1. Start from a dry (snap-frozen) cell pellet
SECTION: Isolate nuclei
2. Add hypotonic lysis buffer (HLB) to cell pellet: 200 µL for 1x106 cells and resuspend softly 
 

SECTION: Isolate nuclei
3. Rotate for 30 min at 4°C to promote lysis of cell membrane (mechanical shear)
SECTION: Isolate nuclei
4. Pellet the nuclei by centrifugation at 10.000g for 10 min at 4°C
SECTION: Isolate nuclei
5. Discard the supernatant
SECTION: Extract histones
6. Resuspend the pellet in 0.4N hydochloric acid (HCl) by soft pipetting until no clumps left in solution by adding 125 µL HCl for 1x106 cells (if necessary: vortex) 
SECTION: Extract histones
7. Incubate for 30 min in acid on rotator at 4°C to promote lysis of nuclei and solubilization of histones
SECTION: Extract histones
8. Spin down for 10 min at 4°C and 16.000 g
SECTION: Extract histones
9. Transfer supernatant to new Eppendorf (histones are present in the acid since they are alkaline proteins)
SECTION: Isolate histones
10. Add, drop by drop, trichloroacetic acid (TCA) until a final concentration of 33% is reached to promote precipitation of histones and invert the tube several times (results in a milky solution)

SECTION: Isolate histones
11. Incubate on ice for 30min

SECTION: Isolate histones
12. Spin for 10 min at 4°C and 16.000 g to pellet the histones

SECTION: Isolate histones
13. Remove the supernatant 
 

SECTION: Wash steps
14. Add ice-cold acetone (do not resuspend the pellet) to remove TCA, make sure the pellet is fully covered with acetone

SECTION: Wash steps
15. Spin for 5 min at 4°C and 16000 g

SECTION: Wash steps
16. Remove the supernatant

SECTION: Wash steps
17. Add cold acetone again (do not resuspend the pellet) to remove TCA

SECTION: Wash steps
18. Spin for 5 min at 4°C and 16000 g

SECTION: Wash steps
19. Remove the supernatant
SECTION: Preparation for SDS-PAGE
21. Resuspend in MilliQ water (50 µl for 1x106 cells)

SECTION: Preparation for SDS-PAGE
22. Transfer 400.000 cells to a new Eppendorf tube for gel-electrophoresis (optionally)

 

SECTION: Preparation for SDS-PAGE
23. Vacuum dry the samples (SpeedVac)
 

SECTION: Wash steps
20. Dry at room temperature for 30 min (until no acetone left)
TITLE: How to import OAI-PMH identifiers in OpenRefine
AUTHORS: Andrea   Marchitelli, Alessandra Moi, Carlo Bianchini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocollo per importare in OpenRefine una lista di identificatori OAI-PMH per successive elaborazioni.</div></div>

[STEPS]
?. Open OpenRefine and choose "Create project".
?. Select "Web Addresses (URLs)" as the data source for the new project and click "Next". Enter the OAI-PMH baseurl of the identified target, choose the ListIdentifiers verb and the appropriate metadataPrefix.E.g. http://www.bibliotecheoggi.it/trends/oai?verb=ListIdentifiers&metadataPrefix=oai_dc
The OAI-PMH protocol allows per-date and per-set limiters to be set to expose partial datasets and allow incremental collections.Es. http://an.oa.org/OAI-script?verb=ListIdentifiers&from=1998-01-15&metadataPrefix=oldArXiv&set=physics:hep
?. Select "XML Files" in the "Parse Data As" section.
?. In the preview area of the file to be imported, locate the first line containing the  element and bring the mouse pointer to highlight it.Then click on it.
?. At that point you will have obtained the list of OAI-PMH identifiers, one per line, ready for further processing.
TITLE: Protocol for Preparing SOC
AUTHORS: Prince Samoh

[STEPS]
?. Total Volume = 100ml
?. Weigh 2g of Tryptone into a 150 ml beakerAdd 0.5g of Yeast ExtractAdd 0.058g of NaCl [10mM]Add 0.019g KCl [2.5mM]Add 1mL 2M MgCl2 [10mM]Add 0.12g MgSO4 [10mM]Top up with distilled water to reach the 100ml mark
?. Autoclave solution at 121oC, 0.1MPa for 15 -20 minutes
?. Allow the solution to cool to about 50oC  Add 0.396g of Glucose [20mM]
?. Sterilize the final solution by passing it through a 0.2 µm filter.SOC medium can be stored at room temperature and is stable for several years.
TITLE: T cell isolation from mouse tissues
AUTHORS: Kristin Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps for isolating murine T cells from mouse blood, ascites, spleen, and ID8 tumors.</div><div class = "text-block"><span>In brief: Spleens were disrupted by mechanical force through a 70mm filter (BD Falcon, ref: 352350) and red blood cells lysed using ACK lysis buffer (Gibco, cat: A1049201). Ovarian tumors were excised and normal adjacent tissue removed. Tumors were minced using a GentleMACS dissociator (Miltenyi), incubated for 10 minutes on a rotator (Miltenyi) at 37°C (medium spin setting), disrupted through a 70mm filter, washed with RPMI containing 5% FBS, and lymphocytes were purified on a 44/67% Percoll gradient (800</span><span style = "font-style:italic;">xg</span><span> at 23°C for 20 minutes; GE Healthcare, cat: 17-0891-01).</span></div></div>

[STEPS]
?. [Spleen digestion]
1. Harvest cells by smashing spleen through a 70um filter.2. Spin down cells at 369xg for 4-5 minutes at 4C.3. Remove supernatant and lyse red blood cells with 2 mL ACK lysis buffer for 2 minutes at room temperature.4. Quench lysis using 10mL T cell media.5. Spin down cells at 369xg for 4-5 minutes at 4C. 6. Remove supernatant and resuspend cells in T cell media. Store on ice until use.
?. [Blood and Ascites digestion]
1. Spin down cells at 369xg for 4-5 minutes at 4C.2. Remove supernatant and lyse red blood cells with 2 mL ACK lysis buffer for 2 minutes at room temperature.3. Quench lysis using 2mL T cell media. Spin down cells at 369xg for 4-5 minutes at 4C. 4. Remove supernatant and lyse red blood cells a second time with 2 mL ACK lysis buffer for 2 minutes at room temperature.5. Quench lysis using 2mL T cell media. Spin down cells at 369xg for 4-5 minutes at 4C. 6. Remove supernatant and resuspend cells in T cell media. Store on ice until use.
?. [ID8 ovary tumor digestion]
1. Excise tumor from animal and remove normal tissue. Be sure to remove any lymph nodes from tumor! Store in RPMI containing 5% FBS on ice until processing.2. Transfer tumor tissue into 3mL RPMI+5%FBS in a GentleMACs C tube.3. Mince tumors using a GentleMACS dissociator (Miltenyi). Use protocol 'm_imptumor_01'.4. Transfer tissue to a 50 mL conical tube. Add 35 mL of collagenase (pre-warmed to 37C).5. Put tubes on a MACS Mix Rotator and place in the 37C incubator rotating on medium speed for 10 minutes.6. Spin down tubes at 369xg for 4-5 minutes at 4C. 7. Remove supernatant and resuspend tissue in 5mL RPMI+5%FBS. Smash sample through a 70um filter.8. Spin down tubes at 369xg for 4-5 minutes at 4C. 9. Resuspend pellet in 5mL of 44% Percoll.10. Underlay with 3-5mL of 67% Percoll.11. Spin samples for 20 minutes at 809xg at room temperature with NO BRAKE.12. Remove and discard fat layer floating on top. Gently remove the cells at the interface and transfer to a fresh tube.13. Wash cells with 15mL RPMI+5% FBS. Spin cells at 518xg for 10 minutes at 4C.14. Remove supernatant. Resuspend cells in either RPMI+5% FBS and store on ice until use, or proceed directly to cell staining for FACS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the quantification of luciferase activity in Ostreococcus and Bathycoccus cells that have been transformed with a firefly luciferase  coding sequence upstream of the OTTH595 high affinity transporter promoter. Using this methods we were able to show that O. tauri promoters work in Ostreococcus RCC809 and Bathycoccus.  </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Preparation of 1L of Foraging Buffer
AUTHORS: Alfonso  Pérez Escudero, Alid Al-Asmar, gabrielmadirolas

[DESCRIPTION]
Protocol is mainly inspired from: WormBook Methods http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html.
We use this protocol for our foraging experiments with the worm Caenorhabditis elegans, using different strains of bacteria.
This protocol describes how to produce the buffer we use to wash our bacteria after growing them in LB. The buffer has a similar composition as our Foraging Medium, but contains no cholesterol. It contains antibiotics at bacteriostatic concentrations (for our bacterial strains), in order to prevent bacteria from multiplying after they have been washed.

[STEPS]
1. In order to perform this protocol, you will need the following solutions:
pH=6 phosphate buffer (as prepared in: https://www.protocols.io/view/preparation-of-0-5l-of-phosphate-buffer-ph-6-0-n2bvj8r7bgk5/v1)
1 M magnesium sulfate (as prepared in: https://www.protocols.io/view/preparation-of-1m-magnesium-sulfate-solution-mgso4-ca4rsgv6)
1 M calcium chloride (as prepared in: https://www.protocols.io/view/preparation-of-1m-calcium-chloride-solution-cacl2-b8parvie)
10 mg/mL chloramphenicol in absolute ethanol (stored in freezer)
50 mg/mL novobiocin in milliQ water (filter-sterilized and then stored in freezer, in 1.8 mL aliquots to avoid defreezing-refreezing the whole stock)
2. Add 2.32 g of sodium chloride (NaCl). 
3. Add 780 mL of milliQ water.
4. Add a clean magnet (for stirring) and autoclave.
5. Put the bottle on a stirrer, and wait until it cools down to around 60°C. At this temperature, the bottle should feel very hot to the touch, but one can hold it for a while without feeling too uncomfortable. The agar should still be liquid.
6. While the mix is being stirred, do the following steps in that order, dispensing the reagents right above the surface (to avoid bubbles) with a sterile pipette: 
7. Add 20 mL of phosphate buffer as prepared in https://www.protocols.io/view/preparation-of-0-5l-of-phosphate-buffer-ph-6-0-n2bvj8r7bgk5/v1. This should be added first to prevent any unnecessary precipitation of the next reagents. Wait for ~20 seconds after adding the buffer to give it time to mix well and stabilize the pH.
8. Add 0.8 mL of 1M magnesium sulfate (MgSO4) solution as prepared in https://www.protocols.io/view/preparation-of-1m-magnesium-sulfate-solution-mgso4-ca4rsgv6. 
10. Add 0.8 mL of chloramphenicol (10 mg/mL in absolute ethanol, stored in freezer).
11. Add 1.6 mL of novobiocin (50 mg/mL in milliQ water, stored in freezer).
9. Add 0.8 mL of 1M calcium chloride (CaCl2) solution as prepared in https://www.protocols.io/view/preparation-of-1m-calcium-chloride-solution-cacl2-b8parvie.
This step should make the medium a bit cloudier, but remain translucent. 
12. We store our buffer at room temperature, usually for 2-3 weeks without problem. After this time, it is very common that salts will start precipitating in the bottom of the bottle, but we sometimes use the buffer for a few more weeks if it shows no crystals.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>These methods accompany:</p>
<p> </p>
<p>Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin</p>
<p> (2016): Supporting data for 'Comparative optical genome analysis of two Pangolin species Manis pentadactyla and Manis javanica'. GigaScience Database.</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;">This protocol is part of the manuscript: </span><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;"><a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></span><strong><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;"><br /></span></strong></p>
<p> </p>
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">We thank the Leulier laboratory for their help in establishing this protocol.</span></p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: H_QTof_PL_v2
AUTHORS: Fang Bian

[DESCRIPTION]
To extract plasma lipids for LCMS analysis.

[BEFORE_START]
Use LCMS grade water and solvents. 

[GUIDELINES]
Use glassware throughout this protocol. 

[STEPS]
SECTION: To extract plasma lipids for LCMS study
1. Transfer plasma 50 ul (regular pipette tips) into a 3 ml glass tube
SECTION: To extract plasma lipids for LCMS study
2.  Add 200 ul of LCMS grade-chloroform/methanol (2:1)
SECTION: To extract plasma lipids for LCMS study
3. Stand for 5 min at room temperature.
SECTION: To extract plasma lipids for LCMS study
4. Vortex for 1 min
SECTION: To extract plasma lipids for LCMS study
7. Repeat steps 2-5
SECTION: To extract plasma lipids for LCMS study
5. Centrifuge 12,000 g, for 5 min at 4c
SECTION: To extract plasma lipids for LCMS study
8. Use glass pipette tips to collect the lower organic phase and transfer the same tube in step 6.
SECTION: To extract plasma lipids for LCMS study
9. Evaporate the extracted solvents to dryness 
SECTION: To extract plasma lipids for LCMS study
10. Cap the tube, store at -20oC
SECTION: To extract plasma lipids for LCMS study
11. Deliver the sample to mass spec core.
SECTION: To extract plasma lipids for LCMS study
6. Use glass pipette tips to collect the lower organic phase and transfer the liquid to a new glass tube.
TITLE: Preparation of 1000X Antibiotic Stock Solution
AUTHORS: NUS iGEM

[STEPS]
?. Weigh x grams of antibiotic-of-interest.
?. Dilute in pure 100% ethanol for chloramphenicol or sterile deionized water for kanamycin and ampicillin.
?. Syringe filter water-based antibiotic solution using a 0.22-μm filter
TITLE: Fluorescent Western Protocol
AUTHORS: Lynn   Doran, Steven J Burgess

[DESCRIPTION]
Analysis of proteins using fluorescent immunoblot.  

Note:  
- The choice of secondary antibody depends on the choice of primary antibody, whether it is derived from a mouse (monoclonal) or a rabbit (polyclonal).  
- It is advisable to stick to the 800CW wavelength to avoid problems with chlorophyll autofluorescence encountered with the 680CW antibodies.  

Literature: 
Licor's "Fluorescent Western Blot Detection"
Licor's "Good Westerns Gone Bad"

[BEFORE_START]
Isolate total protein via Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco).
Quantify protein via Protein Concentration Determination using Qubit 4 Fluorometer.
Separate protein components via SDS-PAGE gel electrophoresis.
Transfer protein to a membrane via Protein Transfer using Bio-rad TransBlot Turbo.

[STEPS]
1. 
Keep membranes in the black Western Blot incubation box for all steps, this is important after adding the secondary antibody because the signal is light-sensitive and will become bleached if exposed to light for a long enough period.

 


2. Wet with 1x PBS for 2 min min

3. Rinse membrane with dH2O
4. Discard PBS and incubate with   60 min  at Room temperature
 



6. Incubate with primary antibody (appropriate dilution in    at 4 °C    (in cold room) with gentle agitation on a platform shaker   .
 


5. Prepare 1 L PBS-T solution by diluting 100 mL of 10X PBS

7. Pour off the primary antibody and rinse the membrane with PBS-T.
 

8. Cover the membrane with PBS-T, shake vigorously on a platform shaker at  . Repeat 3 times.

 


9. Create a working dilution of secondary antibody using . . For PVDF membranes only, add 0.01% SDS to the antibody diluent.    Microcentrifuge secondary antibody and pipette from supernatant to precipitate out any protein complexes that may have formed during storage.  

 

 

10. Incubate for 60 min Room temperature with gentle agitation on a platform shaker.
 

11. Pour off the secondary antibody and rinse membrane with distilled water to remove residual blocking agent.
12. Cover the membrane with PBS-T, agitate  
 

13. Discard PBS-T. Repeat step 12  three times.
 

14. Rinse then cover the membrane with 1x PBS,  . 

15. Proceed to imaging blot on LI-COR Odyssey CLx imaging system.
TITLE: p1 11/5 
AUTHORS: Maria Guliakina

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

[STEPS]
?. test
?. {"blocks":[{"key":"ccqb3","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}