eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol for Making Soil
AUTHORS: Yuriani Palomino, Karma Terlik

[DESCRIPTION]
This protocol outlines 3 soil recipes for their corresponding plant species: Phacelia parryi, P. campanularia, and Brassica rapa. In addition to the recipes, it also details the soil mixing process.

[STEPS]
SECTION: Soil Recipe for Phacelia campanularia
1. 1 part peat moss
0.5 part vermiculite
1.5 part sand
3 parts perlite
3 tbsp of fertilizer per 2 gallons of soil
SECTION: Soil Recipe for Brassica rapa
2. 1 part peat moss
1 part vermiculite
1 part perlite
3 tbsp of fertilizer per 2 gallons of soil
SECTION: Soil Recipe for Phacelia parryi
3. 1 part peat moss
1 part vermiculite
1 part sand
2 part perlite
3 tbsp of fertilizer per 2 gallons of soil
SECTION: Soil Mixing
4. Depending on what plant you desire to plant, follow the corresponding soil recipe to measure all required parts except perlite and fertilizer and place all parts within a sterile container.
SECTION: Soil Mixing
5. Using gloved hands or a shovel thoroughly mix the soil until there are not hidden pockets of unmixed material.
SECTION: Soil Mixing
6. If the recipe calls for perlite, add the perlite parts to the container and rinse with DI water to minimize dust. Once wet, repeat step 5.
SECTION: Soil Mixing
7. Depending on the total number of gallons of soil produced, measure out the correct amount of fertilizer and add it to the soil container. Then, repeat step 5.
SECTION: Soil Mixing
8. Once evenly mixed, your soil is now ready to be used for seed planting and transplanting.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Mouse kidney tissues were harvested, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. All sections (3 micrometers in thickness) were stained with hematoxylin and eosin (H&amp;E), periodic acid-Schiff (PAS, Thermo Scientific) or Masson’s trichrome staining (MTS, Sigma) according to the standard procedures or manufacturer’s instructions. For transmission electro micrographs (EM), kidneys were fixed in Karnovsky’s fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1% cacodylate buffer, pH 7.2 for 2 hrs at room temperature, followed by postfixation with 1% osmunim tetraoxide in cacodylate buffer for 90 min at 4˚C and mordanting with 1% tannic acid in cacodylate buffer.. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined with a Hitachi H-7600 transmission electron microscope located in the Imaging Core Facility of the Oklahoma Medical Research Foundation. We followed previously described histological scoring methods (33). </p>

[STEPS]
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TITLE: RNA extraction from E. coli
AUTHORS: An.Huang

[DESCRIPTION]
RNA extraction is a fundamental step in multiple experiments, for example, qPCR. This protocol helps conduct a simple RNA extraction procedure.

[STEPS]
SECTION: Preparation for experiment
1. Grow an overnight bacterial culture in the appropriate media at an appropriate temperature.
SECTION: Preparation for experiment
2. In the following day, take 1 mLfrom overnight culture and add into 10 mL LB media. Grow until the OD600 reads at 0.6-1.0.
SECTION: RNA extraction
3. Harvest 1.5 mL culture (< 5x10%) by centrifugation at 3.000 rpm, 10 min for 10 min in a 1.5 mL microcentrifuge tube.

SECTION: RNA extraction
4. Discard all supernatant.

 

SECTION: RNA extraction
5. Resuspend the pellet in100 µLfreshly prepared Elution Buffer (10mM Tris-HCL pH 8.5) containing lysozyme (0.4 mg/mL lysozyme for Gram negative bacteria). Mix by tapping gently.


SECTION: RNA extraction
6. Incubate the resuspended pellet at room temperature for 3-5 min for Gram-negative bacteria.
SECTION: RNA extraction
7. Add 400 μL Buffer LY. Mix gently.
SECTION: RNA extraction
8. Transfer the cleared lysate to a DNA Clearance column pre-inserted in a 2 mL Collection Tube. Centrifuge at 13.000 rpm, 2 min. Discard the DNA Clearance column and save the flow-through.

SECTION: RNA extraction
9. Transfer flow-through to the RNA binding column. Add 0.5 volume 100% ethanol to the lysate.
 

SECTION: RNA extraction
10. Centrifuge at 13000 rpm, 1 min. Discard the collection tube with the flow through and put the column back to a new collection tube.

SECTION: RNA extraction
11. Add 500 µL Buffer RB to the column and centrifuge at 13.000 rpm, 30 s. Discard the flow-through.

SECTION: RNA extraction
12. Add another 500 µL RNA Wash Buffer to the column and centrifuge at 13000 rpm, 30 s. Discard the flow-through.
 



SECTION: RNA extraction
13. Add another 500 µL RNA Wash Buffer to the column and centrifuge at 13000 rpm, 30 s. Discard the flow-through and collection tube, put the column into a new collection tube.

SECTION: RNA extraction
14. Centrifuge the column at 13000 rpm, with the lid open, for another 1 min. 

SECTION: RNA extraction
15. Place the column to a RNase-free 1.5 mL tube, add 50-100 μL DEPC treated ddH2O to the column and centrifuge at 13000 rpm, 2 min.
 


SECTION: RNA extraction
16. Store the RNA solution at -20 °C.
TITLE: ImmunoFACS
AUTHORS: Florian  Noack, Silvia Vangelisti, Boyan  Bonev

[DESCRIPTION]
Protocol to isolate distinct cell populations by immunostaining followed by FAC-sorting. 

[STEPS]
2. Add freshly prepared 2% Formaldehyde (from a new vial) in PBS to a final concentration of 1%  and incubate for 10 minutes at room temperature with slow! rotation
3. Add 2M glycine solution to a final concentration of 0.2M to quench the reaction and incubate 5 minutes at room temperature with slow! rotation
4. Centrifuge cells for 5 minutes at 500xg at 4C.
5. Wash with cold PBS with 1% BSA.
6. Resuspend in Wash Buffer.
7. Wash for 10 minutes in the cold room, on the rocker.
8. Split aliquot for negative control – wash again (5 minutes).
9. Spin at 2500xg for 3 minutes at 4˚C.
10. Remove supernatant and add 100µl per 1 million cells of Stain Buffer with primary antibodies. Incubate 1 hour on the rocker in the cold room.
11. Wash cells 2x5 minutes in Wash Buffer and pellet with 2500xg for 3 minutes @ 4˚C.
12. Resuspend in DAPI Buffer and incubate for 10-15 minutes in the cold room. Wash with Resuspension Buffer.
13. Resuspend the cells in Resuspension Buffer at a concentration of ~10 million cells per ml*, pass the cell suspension through a 40µm cell strainer and proceed immediately to FACS-sorting (check staining on slides to test the protocol)

* the optimal concentration depends on the used FACS
14. Sort the cells into ~20µl of cold PBS with 1% BSA. Sorting should not exceed more than 3-4 hours. Wash the sorting tubes with PBS with 1% BSA beforehand to prevent cells from sticking to the sides.
15. After sorting cells can be used immediately or pelleted (5 minutes at 2500xg at 4˚C) and snap-frozen in liquid nitrogen for storage at -80˚C.
1. Prepare a single cell solution using the Milteny Dissociation Kit. Count cells and resuspend in PBS to a concentration of 1 million cells per milliliter.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The plasmids were constructed using standard methods; all sequences were verified by appropriate restriction digestion and/or sequencing. Human full-length <em>IGFBP2</em> cDNA from ASCs fused to a M2-Flag tag was produced with a standard PCR protocol. This sequence (Flag-<em>IGFBP2</em>) was subcloned into the pQCXIN retroviral vector with AgeI and BglII restriction sites. For viral infections, MSCs were plated overnight, then infected with retroviruses in the presence of polybrene (6 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 12 h. After 48 h, infected cells were selected with 600 µg/mL G418 for 10 days.</p>

[STEPS]
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TITLE: Vulture Rehabilitation Manual
AUTHORS: Kerri Wolter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Every single vulture species is under threat, not only in southern Africa, but across Africa and the globe. The dangers to the species vary by location, with most threats being anthropogenic. </div><div class = "text-block">The principle behind this manual is that every vulture’s life counts. Each individual deserves the chance to fly free and reproduce. </div><div class = "text-block">This manual is based on 15 years of VulPro’s experience in rehabilitating African vultures. VulPro’s rehabilitation experience is mainly based on the Gyps species, namely the African White-backed and Cape Vulture; but also with Lappet-faced, White-headed, and Palm-nut Vultures. VulPro also rehabilitates large raptors such as owls, eagles, hawks and secretary birds. Vultures, however, are unique in several respects and therefore warrant their own attention with regards to husbandry and medical treatment. This document is designed to advise and aid the rehabilitation of large, Old World vulture species (namely the Gyps, Torgos, and Trigonoceps species) but should also be considered in the treatment of smaller vulture species. </div></div>

[STEPS]
TITLE: Vesicle Fusion on SiO2 Substrates
AUTHORS: Nicole Voce, Paul Stevenson

[DESCRIPTION]
This protocol outlines the steps to produce large area, uniform supported lipid bilayers on SiO2 substrates with or without patterned features.

[STEPS]
SECTION: Substrate cleaning
1. Clean plain SiO2 or TiO2-patterned substrates by sonicating them in a 1:1 IPA:Acetone mixture for ~2 minutes
Dry with a steady stream of N2
SECTION: Substrate cleaning
2. Oxygen plasma clean the substrates for 2 minutes to remove any residual surface contamination
 

SECTION: Vesicle prep
3. Prepare a 1.2 mg/mL DLPC 0.5mol% Rho-PE solution (suitable for FRAP measurements and general fluorescence imaging) or a 0.3mg/mL DLPC 0.0025mol% Rho-PE solution (suitable for FCS measurements)
SECTION: Vesicle prep
3.1. To prepare 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solutions:

Measure out appropriate amount of DLPC (stored as powder) into a glass vial
 

Add appropriate amount of Rho-PE (stored as 1 mg/mL in chloroform) to DLPC
 

Add 100-200 μL chloroform to vial
  
Dry with N2 so the solution doesn't look glassy-- it should have a dull appearance 

  
Leave in vacuum desiccator overnight to fully remove residual chloroform.




SECTION: Vesicle prep
3.2. After drying, re-hydrate solution with phosphate buffered saline to achieve desired concentrations of 1.2 mg/mL or 0.3 mg/mL 

 

    

SECTION: Vesicle prep
4. Extrude 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution 21 times
 
        

SECTION: Vesicle prep
5. After extrusion, aliquot the solution into Eppendorf tubes and store at -20ºC until ready to use
SECTION: Bilayer formation
6. 
 

Set hotplate to 30ºC
Use double-sided kapton tape to adhere a substrate to the bottom of an imaging chamber

  

SECTION: Bilayer formation
7. Place the substrate on the hotplate and deposit 30 μL of 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution and 90 μL phosphate buffered saline onto the substrate
Cover the substrate with a pipette box top or something else to protect from dust
Let the substrate incubate for 10 minutes

  

SECTION: Bilayer formation
8. After 10 minutes of incubation, deposit 700 μL of phosphate buffered saline onto the substrate, flooding the imaging chamber
  

SECTION: Bilayer formation
9. Wash the substrate by pipetting 500 μL off and then depositing it back on, moving around the substrate
Repeat 15 times
 

SECTION: Bilayer formation
10. After 15 washing steps, take 500 μL off and depose of it

SECTION: Bilayer formation
11. Roll a coverslip over the imaging chamber
 
 
    

SECTION: Bilayer formation
12. Image the samples to confirm bilayer formation. A representative image is shown below: black circles are TiO2 features deposited on the surface to inhibit bilayer formation (scale bar is 100um)
TITLE: Marchantia high throughput imaging in multiwell plates
AUTHORS: marta tomaselli, Marius Rebmann, Mihails Delmans

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol allows high throughput imaging of Marchantia gemmae, using a cheap setup made with broadly available lab equipment. It was developed from the 3D-printed Imaging Growth Chamber developed by Mihails Delmans in his PhD work (</span><a href="https://github.com/HaseloffLab/MihailsDelmansThesis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/HaseloffLab/MihailsDelmansThesis</span></a><span>).</span></div><div class = "text-block">We used a transparent 384 wells plate filled with 1/2 strength Gamborg B5 media with 1.2% agar and placed a single gemma at the centre of the well. Gene frames and coverslips treated with anti-fog spray were used to cover (seal) the wells. This setup can be paired with automated imaging of samples.</div></div>

[STEPS]
?. [Media preparation]
Prepare 1/2 strength Gamborg media plus vitamin  with 1.2% agar.
?. [Plate set up]
Pour the media into your selected wells, making sure they are filled up to the top and they not contain any air bubble inside.
The number of wells to be filled depends on your requirements/ travelling range of the microscope stage. We usually filled one more row/columns of wells around our samples to reduce media evaporation.
?. [Plate set up]
Let the media solidify and level it with a sterile scalple if it bulges out of the wells.
?. [Plate set up]
Place the gene frames to delimit the wells you are planning to image
?. [Plate set up]
Pick a single gemma from a gemma cup and place it at the centre of a well filled with media.
Cover the plate with a lid to prevent media from evaporating when not adding gemmae.
?. [Plate set up]
Clean the cover glass with 70% EtOH and spray one side with the anti-fog spray.
?. [Plate set up]
Let the anti-fog solution evaporate and remove any residue with a lens tissue moving in one direction to avoid any visual artifacts.
?. [Plate set up]
Place the coverslip on the gene frame, making sure the anti-fog treated side is facing down.
?. [Plate set up]
The plate is now ready for imaging. Gemmae can be grown inside the plate up to 7 days.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the preparation and use of ELISpot 96 well plates for measuring human IFNγ and Granzyme B production by immune cells (notably CD4 and CD8 T cells). Cells harvested from peripheral blood or other organs can be used, stimulated with antigen of interest (usually between 18 to 24h), and per cell production of IFNγ and Granzyme B measured as a ‘spot’ on a plate well membrane (<a href="http://www.immunospot.eu/advantage-of-immunospot-kits.html" target="_blank">http://www.immunospot.eu/advantage-of-immunospot-kits.html</a>).</p>

[STEPS]
TITLE: CHCHD2 T61I Mouse Genotyping
AUTHORS: Szu-Chi Liao, Ken Nakamura

[DESCRIPTION]
To genotype CHCHD2 T61I point mutant mice by qPCR.

[STEPS]
SECTION: Mouse Tail Lysis
1. Materials
Isoflurane
Sanitized scissors
DirectPCR lysis reagent (mouse tail, Viagen Biotech, 102-T) 
Proteinase K – Recombinant, PCR grade (Roche, 03115844001)
Microcentrifuge tubes
Incubator
Water bath
SECTION: Mouse Tail Lysis
2. Anesthetize mice with isoflurane.
SECTION: Mouse Tail Lysis
3. Cut no longer than 2 mm of distal tail from mice.
SECTION: Mouse Tail Lysis
4. Place each tail in a sterile, labeled tube.
SECTION: Mouse Tail Lysis
5. Add 200 μl of DirectPCR lysis reagent.
SECTION: Mouse Tail Lysis
6. Add 10 μl of proteinase K and mix well.
SECTION: Mouse Tail Lysis
7. Incubate the tails at 55°C overnight.
SECTION: Mouse Tail Lysis
8. To deactivate proteinase K, incubate tails for 45 min in an 85°C water bath.
SECTION: Mouse Tail Lysis
9. Store extracted DNA at -20°C until use.
SECTION: qPCR Genotyping
10. Materials
Kapa Probe Fast qPCR Master Mix (2X) kit (Kapa Biosystems, KK4715)
EndPoint-F Primer: TGAAGATGGCCCAGATCTG
EndPoint-R Primer: CTGAAGCCCCCAGTGAT
WT-Probe: (5’ JOE) TTAGATGGCTACCACCGCG (3’ Iowa Black)
MT-Probe: (5’ 6-FAM) TTAGATGGCGATCACCGCG (3’ Iowa Black)
Nuclease-free water
SECTION: qPCR Genotyping
11.  Make Reaction Buffer:
 
 
SECTION: qPCR Genotyping
12. Open SDS 2.4 software, create a new experiment and choose “standard curve”.
SECTION: qPCR Genotyping
13. Load the plate.
SECTION: qPCR Genotyping
14.  Set thermal cycling as follows:
 
 
SECTION: qPCR Genotyping
15. In the Setup tab, add detectors for FAM and JOE signal and start the program.
SECTION: qPCR Genotyping
16. After amplification is done, create a new experiment and choose “allelic discrimination”.
SECTION: qPCR Genotyping
17. Assign the markers which detect FAM and JOE signals.
SECTION: qPCR Genotyping
18. Start “Post Read”.
SECTION: qPCR Genotyping
19. Expected Results:
When plotted WT against MT fluorescence signal, each genotype would show a distinct ratio/slope as below:
TITLE: In vivo BioID protein purification
AUTHORS: Shiyi Wang

[DESCRIPTION]
In vivo BioID protein purification

[STEPS]
1. **Animal Preparation** - Breed genotype-matched animals (wild-type C57BL6 or LRRK2 G2019Ski/ki) to produce single-genotype litters. - For each genotype (WT or G2019S), prepare 6 pups for injection.
2. **AAV Injection** - Inject 1 µL of AAVs carrying Astro-Ezrin-BioID (PHP.eB.GfaABC1D-Ezrin WT-BioID2-HA) or Astro-CYTO-BioID (PHP.eB.GfaABC1D-BioID2-HA) bilaterally into the cortex of P0-P2 mouse pups using a Hamilton syringe. - Monitor pups until they recover on a heating pad.
3. **Biotin Injection** - At P18, P19, and P20, subcutaneously inject biotin at 24 mg/kg to increase biotinylation efficiency.
4. **Tissue Collection** - At P21, remove the cerebral cortices and store at -80°C. - Pool 2 genotype-matched cortices at the time of protein isolation, yielding 3 independent replicates per BioID construct.
5. **Protein Purification** - Lyse each cortex in a buffer containing: - 50 mM Tris/HCl, pH 7.5 - 150 mM NaCl - 1 mM EDTA - Protease inhibitor mixture (Roche) - Phosphatase inhibitor mixture (PhosSTOP, Roche) - Add an equal volume of buffer containing: - 50 mM Tris/HCl, pH 7.5 - 150 mM NaCl - 1 mM EDTA - 0.4% SDS - 2% TritonX-100 - 2% deoxycholate - Protease inhibitor mixture - Phosphatase inhibitor mixture
6. **Sonication and Centrifugation** - Sonicate samples. - Centrifuge at 15,000 g for 10 minutes. - Ultracentrifuge the supernatant at 100,000 g for 30 minutes at 4°C.
7. **Sample Preparation for Protein Binding** - Add SDS detergent to the samples and heat at 45°C for 45 minutes. - Cool on ice.
8. **Protein Binding** - Incubate each sample with High-Capacity Streptavidin Agarose beads (ThermoFisher) at 4°C overnight.
9. **Bead Washing** - Wash beads serially:
10. Twice with a solution containing 2% SDS.
11. Twice with a buffer containing 1% TritonX-100, 1% deoxycholate, and 25 mM LiCl.
12. Twice with 1 M NaCl.
13. Five times with 50 mM ammonium bicarbonate.
14. **Protein Elution** - Elute biotinylated proteins attached to the agarose beads in a buffer containing: - 125 mM Tris/HCl, pH 6.8 - 4% SDS - 0.2% β-mercaptoethanol - 20% glycerol - 3 mM biotin - Heat at 60°C for 15 minutes.
15. **Downstream Analysis** - Subject the 12 total samples (3 per genotype per construct) to LC-MS/MS and downstream analysis.
TITLE: Twist 96-Plex (Riptide) Library Prep 
AUTHORS: Katarina A Cohen, Khai-Minh H Nguyen, Oksana Polesskaya, Abraham Palmer

[DESCRIPTION]
This protocol is designed for Twist 96-PLex Library Prep. We use the EPmotion 5075 to add sample barcodes (can also easily be done manually with a multichannel pipette). This is a continuation of the "EPMotion - Normalization and Randomization" protocol. 

[GUIDELINES]
It is optional to use the EPmotion in this protocol. Visit Twist Bioscience site for updated protocols. 

[STEPS]
SECTION: Adding Primer A with EPmotion
2. Fill ice pan with ice chips. 
SECTION: Adding Primer A with EPmotion
3. Defrost 4uL randomized/normalized sample plate from the "EPMotion - Normalization and Randomization" protocol on ice 

SECTION: Adding Primer A with EPmotion
5. Download and import   and   
Depending on the GC content of the species you are working with, choose the protocol accordingly. 
Use 50% GC Content Adapters if samples have GC content between 40% - 60% 
Use Low GC Primers if samples have GC content below 40%
Use High GC Primers if samples have GC content above 60% 

SECTION: Adding Primer A with EPmotion
4. Open epBlue application
SECTION: Adding Primer A with EPmotion
1. Review the Twist 96-Plex Protocol 
Note: Protocol was created for Document Version (DOC-001284 REV 2.0)
Twist Bioscience Protocol

SECTION: Adding Primer A with EPmotion
6. Set up the EPmotion as shown on the worktable below 
    

SECTION: Library Prep
7. Follow the Twist 96-Plex Protocol 
Note: Protocol was created for Document Version (DOC-001284 REV 2.0)
IMPORTANT: Size selection Bead Volumes used: 50ul:20ul (Option 3) worked best for us. 
Make sure to record which pool barcode index for each library
PRO TIP: We try to match the last digit of the library number (Ex. Riptide 53) with the pool barcode index used. 
In the case of Riptide 53. We would use the pool barcode #3 for this particular library. 


SECTION: QC
8. Perform on each library and record keep all QC for pooling steps 
Nanodrop 
Qubit (HS Assay)
Tapestation (D1000) 

SECTION: QC
9. Libraries should have an average fragment size between 420bp - 650bp.
260/280 should be around 1.80 - 2 
260/230  should be around 2-2.2.
We have been able to get good data from libraries with relatively poor nanodrop purities.
Qubit concentrations can widely range. We get a range from 10ng/ul - 60ng/ul
TITLE: Ambulatory Testing - UroMOCA and ColoMOCA
AUTHORS: Brett Hanzlicek, Margot Damsaser, Dennis Bourbeau

[DESCRIPTION]
Ambulatory Recordings of Wireless Bladder (UroMOCA) and Colon (ColoMOCA) Devices in Pigs.


[STEPS]
SECTION: Ambulatory Testing
1. Place BluMOCA receiver radio into pocket on the pig jacket.
SECTION: Ambulatory Testing
2. Wake the UroMOCA and ColoMOCA with the waker set between 9V-15V, 1.5A.
SECTION: Ambulatory Testing
4. Plug the Bluetooth dongle into the computer.
SECTION: Ambulatory Testing
5. Record from from the UroMOCA and ColoMOCA using LabVIEW software and the SD card that is onboard the receiver radio; take notes about pig movement, eating, voiding, bowl movements etc.
SECTION: Ambulatory Testing
6. Turn off UroMOCA and ColoMOCA with waker.
SECTION: Ambulatory Testing
7. Remove BluMOCA receiver.
SECTION: Ambulatory Testing
3. Turn on and place accelerometer into a pocket on the pig jacket.
SECTION: Ambulatory Testing
8. Remove accelerometer.
TITLE: DNA extraction (Salting out)
AUTHORS: Karen Guimaraes

[BEFORE_START]
 In microtubes containing tissue fragments, add 440μL of lysis buffer (10mM Tris-HCl, 2mM EDTA, 400mM NaCl, 2% SDS) and 10 µL of proteinase K (10mg/mL);
 Incubate in a water bath at 55°C for approximately 1:30h or overnight;
 Add 300µL of NaCl (5M) and centrifuge for 10min at 10.000rpm.
 Transfer supernatant containing the DNA to microcentrifuge tube (1,5mL);
 Add 500µl of 100% isopropanol. 
 Centrifuge for 10min at 10.000rpm;
 Discard the supernatant and reuse microcentrifuge tube;
 Add 700µL of 70% ethanol;
 Centrifuge for 3min at 13.000rpm;
 Discard the supernatant and dry microcentrifuge tube;
 Add sterile H2O and 5µL of RNAse (10mg/mL);
 Incubate at 37°C for 30 min and stored at -20ºC. 

[STEPS]