eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is a modified version of the MoBio PowerPlant Pro DNA extraction kit for plant and lichen tissues that have been stored in CTAB buffer.  Prior to being placed in CTAB, photosynthetic tissues from plants and lichens were surface-sterilized with sequential washes in 95% EtOH, 0.5% sodium hypochlorite, and 70% EtOH and air dried under sterile conditions.  Twenty-four tissue pieces per host were placed in 750 uL of CTAB buffer.  Four tubes per host individual were sampled for a total of 96 tissue pieces. </p>
<p><strong> </strong></p>

[BEFORE_START]
<div class="page" title="Page 1">
<div class="section" style="background-color: rgb(100.000000%, 100.000000%, 100.000000%);">
<div class="layoutArea">
<div class="column">
<p><strong>You will need the following reagents and equipment for the protocol. </strong></p>
<p><span style="font-size: 11.000000pt;"> </span></p>
</div>
</div>
</div>
</div>
<p><strong>Reagents:</strong></p>
<p><em>-DNase Away</em></p>
<p><em>-Stainless Steel Beads, 3.2 mm (Next Advance Cat # SSB32)</em></p>
<p><em>-Stainless Steel Bead blend, 0.9-2.0mm (NextAdvance Cat # SSB14B)</em></p>
<p><em>-Zirconium Oxide Beads, 2.0 mm (Next Advance Cat # ZRoB20)</em></p>
<p><em>-Liquid Nitrogen</em></p>
<p><em>-MoBio PowerPlant Pro DNA extraction kit (MoBio Cat # 13400-50)</em></p>
<p><strong> </strong></p>
<p><strong>Equipment</strong></p>
<p><em>-Laminar flow hood</em></p>
<p><em>-Lyophilizer</em></p>
<p><em>-Centrifuge capable of handling 24 1.5-2.0 mL tubes</em></p>
<p><em>-Optional: PowerVac™ and 40 autoclaved Mini Spin Filter Adapters</em></p>
<p><em>-Next Advance Bullet Blender Storm or comparable bead beater</em></p>
<p><em>-Heat block and water bath at 65C</em></p>
<p><em>-Parafilm</em></p>
<p><em>-Sterile needle</em></p>
<p><em>-1000 uL filter tips</em></p>
<p><em>-200 uL filter tips</em></p>
<p><em>-20 uL filter tips</em></p>
<p><em>-10 uL filter tips</em></p>
<p><em>-Freezer block (-20C)</em></p>

[GUIDELINES]
<p><strong>Guidelines:</strong></p>
<ul>
<li><em>Gloves must be worn at all times. Change gloves frequently and decontaminate often with DNA Away.</em></li>
<li><em>Pipetting must be done extremely carefully to minimize the risk of aerosols that can easily cause contamination between samples. </em></li>
<li><em>Use only unopened, sterile, aerosol-resistant pipette tips (filter tips) to minimize contamination of the pipette shaft and your samples. Clean the body of the pipettes regularly with DNA Away. Discard pipette tips after each use to avoid cross-contamination. Eject pipette tips carefully to prevent aerosol formation or other splashing. </em></li>
<li><em>Remember to always clean the hood and any equipment with DNA Away and decontaminate where possible with UV for 30 minutes prior to use to avoid cross-contamination.</em></li>
<li><em>The pipettes used need to be calibrated annually.</em></li>
<li><em>Be careful not to touch the inside lid of tubes as it will cross-contaminate samples. </em></li>
</ul>
<p><em> </em></p>

[STEPS]
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TITLE: First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol explains methods for the "First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)".

[STEPS]
1. In a sterile microfuge tube add:
 

3. Add the following (bringing to 20 µl final volume):
 

5. Incubate at 42 °C for 30 minutes-1 hour.
6. Inactivate enzyme at 80 °C for 5 min.
7. Store products at -20 °C or proceed to next step(s).
2. Optional: Heat for 3-5 minutes at 65 °C-70 °C. Spin briefly and place promptly on ice.
4. If Random Primer Mix is used, an incubation step at 25 °C for 5 min is recommended before the 42 °C incubation in the next step.
TITLE: Schedule of Study Visits and Evaluations (Appendix A of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Appendix A of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: Semi-automated tracing of hamstring muscle architecture for B-mode ultrasound images.
AUTHORS: Kevin Cronin, Eamonn Delahunt, Shane Foley, Sean Cournane

[DESCRIPTION]
Hamstring strains are the most prevalent injury sustained by field-sport athletes. Insufficiencies in the architectural characteristics of the hamstring muscles can heighten an athlete’s risk of incurring a hamstring strain. To evaluate the influence of hamstring muscle architectural characteristics (i.e., fascicle length, pennation angle, muscle thickness) on injury risk, it is necessary to precisely evaluate these characteristics. Considering this, our aim was to develop and evaluate the precision of a novel semi-automated tracing software to measure the architectural characteristics of the biceps femoris long head (the most commonly injured hamstring muscle) in B-mode ultrasound images.We acquired static sonograms of the biceps femoris long head from ten healthy male field-sport athletes. The architectural characteristics (fascicle length, pennation angle and muscle thickness) of participants’ biceps femoris long head were evaluated 10 times using the tracing software, with the specific purpose of determining its measurement precision.

[STEPS]
TITLE: Find  proteins of unknown function (PUF) using Plantannot - Protocol C 
AUTHORS: Marcos Viana, Adhemar Zerlotini, Mauricio Mudadu

[STEPS]
?. [Entering application]
Enter the Plantannot Result's page, with empty filters and text box search: https://www.machado.cnptia.embrapa.br/plantannot/find/?q=Or you can enter the  https://www.machado.cnptia.embrapa.br/plantannot  initial page and click on the magnifying glass with the text box empty as well.
?. [Filtering]
Visualize the "Filters" card on the left of the page from step1:
?. After execution of all filters we will have the following list of filters:
?. [Filtering]
In the "Organisms" filter, select any organisms (expand the organism's list using the green arrow) or select all by leaving all boxes empty. We will use Oropetium tomaeum as example. Click "apply" to execute the filter:
?. [Filtering]
In the "Feature type" filter, select "polypeptide", and click "apply" to execute the filter:
?. [Filtering]
In the Orthology filter, select "orthology", and click "apply" to execute the filter:
?. [Filtering]
Leave the "Coexpression" filter empty:
?. [Filtering]
In the "Orthologs_coexpression" filter, select "co-expression", and click "apply" to execute the filter:
?. [Filtering]
In the "Analyses" filter, select both "no diamond matches" and "no interproscan matches", and click "apply" to execute the filter:
?. [Filtering]
Leave the "Biomaterial" and "Treatment" filters empty:
?. [Viewing results]
Visualize the "Results" card on the center-right of the screen, we will have the resulting list of Oropetium's PUFs supposedly related to abiotic stress by using orthology and co-expression networks:
TITLE: Mouse - Stellate Isolation Protocol
AUTHORS: John Tompkins, Jeffrey Ardell, Kalyanam Shivkumar

[STEPS]
?. Mouse: C57BL/6JSex: M/FAge: 12 ± 2wks
?. Euthanesia:Overdose isoflurane (5%), decapitation.
?. Materials:4x insect pins1x minutien pins1x surgical tools (scissors, toothed forceps, straight forceps, iridectomy scissors)1x foam dissection board (8 x 10 x 1 inch)1x absorbent pad2x petri dish with Sylgard 500 mls ice cold physiological salt solution (PSS, in mM: 121 NaCl, 5.9 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 25 NaHCO3, 2 CaCl2, 8 D-glucose; pH 7.4 maintained by 95% O2-5% CO2 aeration)
?. 1.      Kill mouse with isoflurane overdose (5%), decapitation and exsanguination. 2.       Fix limbs to dissection board with pins.3.       Cut open abdomen with transverse incision below xiphoid process with small scissors.4.       Open diaphragm and remove thorax containing heart and lungs.5.       Place thorax in iced PSS for 10 mins.6.       Pin dorsal surface of thorax to Sylgard floor of petri dish and submerse with PSS.7.       Cut ribs bilaterally moving caudal to rostral to remove ventral thorax.8.       Discard lungs and pin heart to left side.
?. 9.    Locate stellate ganglion under 1st or 2nd rib near costovertebral joint, in close proximity to the spine, next to the subclavian/mammalian artery.  The stellate is connected caudally to the paravertebral ganglia. Follow paravertebral chain down to confirm it’s the stellate ganglion. The stellate ganglion is the first ganglion larger than the others. The two arms (ansa subclavia) that emerge from the stellate ganglion encircle the subclavian artery.
?. 10. Isolate and remove stellate.11.  Pin isolated stellate to Sylgard in clean petri dish.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The scope of the protocol is the quantification of pigments in pasture samples in the context of proximal and remote sensing-based studies. The protocol describes a standard procedure for the destructive sampling of mixed pasture covers for the estimation of biophysical variables (biomass, leaf area index, canopy water content, specific leaf area, specific leaf weight), pigments, and nutrients without discrimination by species or functional types and the laboratory procedure for the samples pretreatment and quantification of pigments contents (Chlorophyll a, Chlorophyll b, and carotenoids)  in the collected samples. The procedure allows the quantification of pigments in herbaceous covers per fresh or dry mass as well as per sampling area. The determination of pigments is performed by a cold extraction procedure with 80% v/v aqueous acetone followed by a spectrophotometric determination.</p>

[GUIDELINES]
<p><strong>PROTOCOL FOR PIGMENTS CONTENT QUANTIFICATION IN HERBACEOUS COVERS: SAMPLING AND ANALYSIS</strong></p>
<p> </p>
<p>Protocol developed in the context of the SynerTGE research project: <em>Landsat-8+Sentinel-2: exploring sensor synergies for monitoring and modelling key vegetation biophysical variables in tree-grass ecosystem </em>(GL-2015-69095-R).</p>
<p> </p>
<p><strong>SCOPE</strong></p>
<p> </p>
<p>Field and laboratory protocols for pigments quantification in herbaceous covers in the context of proximal and remote sensing-based studies.</p>
<p> </p>
<p> </p>
<p><strong>OBJECTIVE</strong></p>
<ol>
<li>Description of a standard procedure for destructive sampling in herbaceous covers for the estimation of biophysical variables (biomass, leaf area index, canopy water content, specific leaf area, specific leaf weight), pigments, and nutrients in green and dry vegetation fractions without discrimination by species or functional types</li>
<li>Description of the laboratory procedure for the quantification of pigments (Chlorophyll a, Chlorophyll b, and carotenoids) contents in the collected samples.</li>
</ol>
<p> </p>
<p><strong>INDEX</strong></p>
<ol>
<li>FIELD SAMPLING PROTOCOL
<ul>
<li>Objective</li>
<li>Materials and equipment</li>
<li>Sampling procedure</li>
</ul>
</li>
<li>LABORATORY SAMPLES PRE-TREATMENT</li>
</ol>
<ul>
<li>Objective</li>
<li>Equipment and Materials</li>
<li>Procedure</li>
</ul>
<ol start="3">
<li>PIGMENT EXTRACTION PROCEDURE
<ul>
<li>Objective</li>
<li>Equipment, materials and reagents</li>
<li>Pigment extraction</li>
<li>Calculations</li>
<li>Quality check</li>
<li>References</li>
</ul>
</li>
</ol>

[STEPS]
?. 
?. 
?.
TITLE: Kings B Media (1L)
AUTHORS: Brian Smith, baltrus@email.arizona.edu micro.tucson

[STEPS]
?. [Fill container aproximately 750mL of H2O]
Add stir bar and set to mix
?. [Add Bacto Peptone]
[Bacto Peptone]
[Bacto Tryptone]
[Glycerol]
?. [Add K2HPO4]
or of 1M Solution
1 g
10 ml
?. [Add MGSO4•H2O]
or of 1M Solution
1 g
10 ml
?. [Bring volume to 1L by adding H2O]
Allow to mix again with stir bar.
?. [Add Agar (if making plates)]
Add agar to container to be autclavedThen add media
[agar]
TITLE: Standard Volvox Medium
AUTHORS: Raymond Goldstein, Kyriacos Leptos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for the execution of a Standard Volvox Medium recipe. Starr's (1969) modification of Provasoli & Pintner's Medium (1959), as further modified by Starr, Kochert and Kirk at various times. </div></div>

[STEPS]
?. [Autoclaving]
Dispense the mixture as 500 ml batches into desired 1L bottles.
?. [Adding Vitamins (Just before use)]
Using pre-plugged sterile tips mix the following as a cocktail in a 1.5 ml sterile microcentrifuge:
Always add vitamins fresh and keep the medium at  .
Be sure to work under a laminar flow hood of this step.
[Thiamine (Vitamin B1)]
[Biotin (Vitamin B7)]
[Cyanocobalamin]
?. [Adding Solvent]
Add  to a 2L beaker.
[Initial MilliQ water]
?. [Adding Macro minerals, N-source, and S-source]
Add .
[Ca(NO3)2]
?. [Adding Macro minerals, N-source, and S-source]
Add .
[MgSO4]
?. [Adding Macro minerals, N-source, and S-source]
Add .
[KCl]
?. [Adding Macro minerals, N-source, and S-source]
Add .
[Na2CO3]
?. [Adding N-source]
Add .
[Urea]
?. [Adding P-source]
Add .
[Na2Glycerophosphate]
?. [Adding pH Regulator]
Add .
[HEPES]
?. [Adding Trace Minerals]
Add .
["P IV Metal solution" 2X]
?. [Adjusting pH and final volume]
Adjust the pH to  with NaOH.
Make sure this is done under mild mixing.
?. [Adjusting pH and final volume]
Adjust the final volume to .
1000 ml
?. [Adding Vitamins (Just before use)]
Add .
[Thiamine (Vitamin B1)]
?. [Adding Vitamins (Just before use)]
Add .
[Biotin (Vitamin B7)]
?. [Adding Vitamins (Just before use)]
Add .
[Cyanocobalamin]
?. [Adding Vitamins (Just before use)]
Mix.
?. [Adding Vitamins (Just before use)]
Add the vitamins to the SVM-vit.
?. [Autoclaving]
Autoclave the medium.
?. [Autoclaving]
Keep the medium at  until you are ready to use it.
4 °C
TITLE: CODEX FFPE Microtome Sectioning
AUTHORS: Joanna Bi

[DESCRIPTION]
Protocol for sectioning FFPE blocks for use with CODEX

[STEPS]
1. Place the paraffin blocks on wet ice for 5-10 minutes to rehydrate
2. Heat up a water bath to 40º C
3. Insert a new blade into the microtome
4. Prepare Superfrost Plus Microsocope slides (Cat. No: 1255015) next to the microtome
5. Place a paraffin block on to the microtome and cut into it until the desired part of the sample is reached
6. Section the tissue at 4 μm

7. Place the sectioned tissue in the water bath until it flattens out
8. Place a prepared slide in the water bath, move it towards the tissue, and gently lift it out of the bath 
8.1. Make sure that the tissue is within these boundaries
 

9. Change the blade (or move it so an unused portion is being used) between samples and when there is paraffin build up or chipping on the blade
10. Let the slides dry facing upwards at an angle
11. Store the slides at 4º C for up to 6 months
TITLE: cDNA synthesis using the QuantiTect Reverse transcription kit
AUTHORS: Enrico Bagnoli, Miratul Muqit

[DESCRIPTION]
This protocol details the synthesis of cDNA.

[STEPS]
SECTION: cDNA synthesis
1. Thaw template RNA on ice. Thaw gDNA wipeout buffer, Quantiscript® Reverse Transcriptase, Quantiscript RT buffer, RT Primer Mix and RNase-free water at room temperature (15 °C25 °C).
 
Mix each solution by flicking the tubes. Centrifuge briefly to collect residual liquid from the sides of the tubes, and then keepon ice .
SECTION: cDNA synthesis
2. Prepare the genomic DNA elimination reaction on ice according to Table 1. Mix and then keep on ice.

 



 

SECTION: cDNA synthesis
3. Incubate for 2 min at 42 °C, then place immediately on ice.
 


SECTION: cDNA synthesis
4. Prepare the reverse-transcription master mix on ice according to Table 2. Mix and then keep on ice. The reverse-transcription master mix contains all components required for first-strand cDNA synthesis except template RNA.

 



 

SECTION: cDNA synthesis
5. Add template RNA from step 3 (14 µL) to each tube containing reverse-transcription master mix. Mix and then store on ice.
SECTION: cDNA synthesis
6. Incubate for 15 min at 42 °C.

 

SECTION: cDNA synthesis
7. Incubate for 3 minat 95 °C to inactivate Quantiscript Reverse Transcriptase.
SECTION: cDNA synthesis
8. Place the reverse-transcription reactions on ice and proceed directly with real-time PCR. For long-term storage, store reverse-transcription reactions at -20 °C.
TITLE: Reverse Transcription of Phage RNA
AUTHORS: Frej   Larsen

[DESCRIPTION]
Reverse transcription (RT) is the process of turning ssDNA into cDNA using the reverse transcriptase enzyme. This protocol uses the RT reaction to turn ssRNA and dsRNA into ssDNA and is specifically used for virome analysis.

[STEPS]
1. Add 16 µL  sample to a PCR tube and denature RNA by heating to 95 °C for 5 min  on a ThermoCycler

2. Centrifuge for 10 s  and put on ice

3. Add 4 µL  of the 5X master mix to the PCR tube, then run the following program on a ThermoCycler:

25 °C for 10 min  (primer annealing)
50 °C for 10 min  (reverse transcription)
80 °C for 5 min  (enzyme inactivation)





4. Centrifuge for 10 s 

5. Purify by bead cleaning and elute in 10 µL MQ water
TITLE: No Title
AUTHORS: 

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

[BEFORE_START]
<div>
<div>
<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 familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>

[STEPS]
?. 
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TITLE: Protocol for dissection and tissue preparation of the spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae) for morphometric analysis and morphological analysis using scanning electron microscopy
AUTHORS: Alina Avanesyan, Timothy K. Maugel [Laboratory for Biological Ultrastructure, William O. Lamp [Department of Entomology

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this study, we focused on assessing changes in morphology of (a) the lanternfly mouthparts (stylets and labium), and (b) the lanternfly tarsal tips (arolia and tarsal claws) at each developmental stage</div></div>

[STEPS]
TITLE: Kelp DNA extractions - HMW 
AUTHORS: wpearman 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">DNA extraction protocol for Durvillaea antarctica with extremely high polysaccharide levels.</div></div>

[STEPS]
?. Add 500mg of freeze dried kelp powder to 7mls of lysis buffer (4% CTAB, 2% PVP 40K, 80mM borax, 250mM EDTA, 20mM tris pH 8) and 20ul proteinase K.
This solution is extremely viscous, it couldn't pipette it - instead I poured it into a 15ml tube.
?. Mix together and lyse overnight at 65 degrees
?. Cool to room temperature and add 7mls of chloroform:isoamyl alchol, mix thoroughly, centrifuge for 20 minutes at 4,000rcf
?. Transfer upper phase to new tube and add 0.5 volumes of 10% CTAB and 0.25 volumes of 500mM borax. Mix thoroughly.
?. Centrifuge for 10 minutes at 4,000rcf and transfer supernatant to new tube (easier to pour it into new tubes than to pipette it).
?. Incubate at 65 degrees for 20 minutes
?. Cool to room temperature and add an equal volume of chloroform isoamyl alcohol. Mix thorougly.
?. Centrifuge for 20 minutes at 4,000 rcf
?. Transfer upper phase to new tube and repeat steps 7-8.
?. To the upperphase, add 1 volume of 100% isopropanol. You may notice that the isopropanol isn't fully miscible in the solution, add 1ml of H2O at a time till the solution is mostly miscible.
?. Incubate on ice for 10 minutes, then centrifuge at 4,000rcf for 5 minutes.
?. Pour off supernatant and add 5mls of 70% Ethanol and mix thoroughly through inversion (use a pipette tip full of 70% ethanol to disoldge the pellet). Centrifuge for 5 minutes at 4,000rcf
?. Repeat step 12
?. Remove supernatant, spin briefly, then remove any residual ethanol.
?. Dry pellet  (I do this using a vacuum centrifuge set to dessicate mode)
?. Add 500ul to 1ml of H2O to 10mM Tris to resuspend
TITLE: Protein Lysate Preparation Protocol (Culture)
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details about the protein lysate of the culture.



[STEPS]
SECTION: Protein Lysate Preparation (Culture)
1. Wash cells using cold PBS followed by ice-cold 1x TNE lysis buffer containing 1% SDS, 0.5% NP-40, 0.5% DOC and protease/phosphatase inhibitors.

TNE lysis buffer:
 

SECTION: Protein Lysate Preparation (Culture)
2. Use cell scraper to remove all detached cells and keep on ice for 5 min.

SECTION: Protein Lysate Preparation (Culture)
3.  Transfer lysates to 1.7 mL collection tubes and sonicate for 2 pulses (at 20% amplitude, 10 seach), heat for 10 min on hot plate (100 °C) and centrifuge at 16000 x g, 10 min, 4 °C.


SECTION: Protein Lysate Preparation (Culture)
4. Transfer resulting supernatants to new tubes (final total lysate).
SECTION: Protein Lysate Preparation (Culture)
5. Perform BCA protein assay following manufacturers instruction and prepare 1xLaemlli samples of standardized concentration for SDS-PAGE.
TITLE: Yale - LDL Cholesterol
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of LDL cholesterol in blood, serum, and plasma. LDL-cholesterol is determined by an elimination method to selectively remove HDL, VLDL, and IDL subfractions prior to assay of LDL-cholesterol as described for total cholesterol. </div></div>

[STEPS]
?. Calibrate Cobas for LDL by running dilutions of the lipid calibrator and the addition of the two direct reagents.
?. Sample handling as performed by the Cobas Mira Plus.     a) Pipette 3 µL of sample into a cuvette slot.    b) Add 225 µL of LDL Direct Reagent 1.    c) Add 75 µL of LDL Direct Reagent 2.    d) Mixture is incubated at 37ºC and spun for 10 minutes.    e) Absorbance is measured at 600 nm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was based on a 2010 paper by Meng and Feldman for extraction of RNA from arabidopsis siliques. While the protocol remains unchanged, I have made some slight edits to streamline it in the lab and also added some notes regarding my results applying this protocol to tobacoo fruit tissue.</p>

[BEFORE_START]
<p>Make up the extraction buffer:</p>
<ul>
<li>100mM Tris-HCl, pH 9.5</li>
<li>150mM NaCl</li>
<li>1.0% sarkosyl</li>
<li>5mM DTT (or 0.5% β-ME)</li>
</ul>
<p>Make up ~3.2mL of 75% ethanol (RNase-free) and at least 200µL 3M sodium acetate (pH 5.2) for each sample to be extracted.</p>
<p> </p>
<p>Set a centrifuge to 4ºC and allow to cool while setting up.</p>

[GUIDELINES]
<p>All solutions should be made with DEPC-treated water and all work should be completed on an RNase-free work surface with RNase-free tools</p>

[STEPS]
?. 
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