eamag/protocols-with-steps huggingface dataset:
TITLE: Anti-condensation agent
AUTHORS: John Bergqvist

[DESCRIPTION]
Preventing plate lids from fogging up by applying an anti-condensation agent. This allows longer recordings of e.g. worm behaviour. 

[BEFORE_START]
Make sure to treat lids that are for use at least a day after treatment. 

[STEPS]
SECTION: Making anti-condensation agent
1. Make a mixture of 0.05% Triton X-100 and 20% ethanol in a 50 mL test tube. 

For 20 mL, mix:
4 mL 
0.1 mL
16 mL
SECTION: Treating plate lids
2. Treating lids with the anti-condensation agent should be done at least a day before using the plates to study worm behaviour. 
SECTION: Treating plate lids
4. Pour the liquid back into the 50 mL tube for later use, or onto new lids to be treated. 
SECTION: Treating plate lids
5. Leave lids to dry for ca 60 min. 

SECTION: Treating plate lids
6. After drying, you will most likely see rings of anti-condensation agent residue on the plate. Wipe these off the lids with Kimberly-Clark Kimwipes until there are no signs of these rings. 
SECTION: Treating plate lids
3. In a Class II Microbiological Safety Hood, pour ca. 3-4 mL of the anti-condensation agent on the inside of the lids and leave for 30 s.

Ensure all of the lid is covered by the anti-condensation agent.
TITLE: Efficacy and safety of the PRO-087 Ophthalmic solution versus systane(R) Ultra and Systane(R) Ultra Preservative-free on the tear film dysfunction syndrome from mild to moderate.
AUTHORS: Sandra Belalcázar-Rey, Valeria Sánchez, Juan C Ochoa-Tabares, Samuel Altamirano, Abraham Soto-Gómez, Ruben Suárez-Velasco, Filiberto García-Félix., Leopoldo Baiza-Durán, Oscar Olvera-Montaño, Patricia Muñoz-Villegas.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The present study was designed to evaluate the efficacy of a sodium hyaluronate/chondroitin sulfate, preservative-free, ophthalmic solution (SH/CS-PF) in normalizing the parameters of the ocular surface, alleviating symptoms and in reducing the squamous metaplasia using the conjunctival impression cytology (CIC) in patients with mild-moderate dry eye disease (DED). </div></div>

[STEPS]
TITLE: Pythium Zoospore Production Soaking Solution
AUTHORS: Nimalka Weerasuriya

[DESCRIPTION]
Creation and test of soaking solutions to be used for large-scale zoospore production for Pythium myriotylum. This is modified from methods in: 
Nyochembeng, L. M., Pacumbaba, R. P., & Beyl, C. A. (2002). Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro. Journal of Phytopathology, 150(7), 396–398. https://doi.org/10.1046/J.1439-0434.2002.00759.X

[STEPS]
SECTION: Soaking Solutions
2. Make Soaking Solutions 1, 2, 3, and Control.
Prep 4 x 1 L autoclavable bottles for each Soaking Solutions (1-3) and Control.

 

SECTION: Preparation
1. Have mature colonies of verified Pythium myriotylum growing on CMA or 1.5-2% WA 90 mm plates. Colony maturity ~7-14 days, with visible oospores.
 

 

SECTION: Soaking Solutions
2.1. Soaking Solution #1, 0.01 Molarity (m) (1 L):
1 L  at pH 7 in 1 L beaker
Add 1 g 
Filter through Whatman #1 filter paper using a funnel into a 1 L bottle.
Add a stir bar to the bottle. 
Adjust pH from pH 8.5  to pH 10.5 to pH 7.0 with  5 N  and  1 N to duplicate the soaking solution in original methods. 
 

SECTION: Soaking Solutions
2.2. Soaking Solution #2, 0.01 Molarity (m)+0.001 Molarity (m) (500 mL):
Transfer 500 mL of SS#1 into a clean 500 mL bottle labeled SS #2.
Add 171 mg to make 0.001 Molarity (m) solution.

 
Check that pH is pH 7.
SECTION: Soaking Solutions
2.3. Soaking Solution #3, 0.001 Molarity (m) (500 mL):
Add 171 mg to make 0.001 Molarity (m) solution in 500 mL  
Check that pH is pH 7.

SECTION: Soaking Solutions
3. Autoclave for 20 minutes on liquid cycle. Remove from autoclave immediately to reduce water evaporation and concentration changes.
 

SECTION: Testing Zoospore Production
4. Take 30 mL of each Soaking Solution   onto surface of Pythium myriotylum plate cultures that have actively produced oospores.

SECTION: Testing Zoospore Production
5. Incubate under light at Room temperature for 1440 min. 
Check for abundant sporangia that will appear after immersion.

SECTION: Testing Zoospore Production
6. After 1.5 up to 4 h every 30 minutes:
Take 80 µL from each immersion to a microcentrifuge tube with 20 µL. Gently invert to stain (or gently vortex) and immobilize zoospores.

SECTION: Testing Zoospore Production
7. Take 10 µL of liquid into haemocytometer and examine under 40x to quantify.
TITLE: GWAS prioritization analysis
AUTHORS: Peter Kilfeather

[DESCRIPTION]
GWAS prioritization analysis from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. A list of 303 genes (sourced from Nalls et al., 2019 supplementary materials),  containing SNPs at an r2 > 0.5 and located within ±1 Mb of 107 common risk variants for sporadic PD was used for prioritization analysis. To convert between human and mouse gene symbols, homologene (v1.4.68.19.3.27, RRID:SCR_002924) and biomaRt (v2.52.0, RRID:SCR_019214) were used. TRAP enrichment (measured as the product of the log­2 fold-change and FDR-adjusted P value) and specificity indices for DAT-TRAP samples were used for gene prioritization, per lead SNP.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<strong>What you need before you start:</strong><br />1. Zobell plates<br />2. Top agar &ndash; 3.5 ml per plate<br />&nbsp; &nbsp; &nbsp; &nbsp;a. MSM<br />&nbsp; &nbsp; &nbsp; &nbsp;b. 6g LMP agarose/liter<br />3. Your host growing somewhere in exponential phase &ndash; 0.3 ml per plate<br />4. Phages &ndash; these could be from:<br />&nbsp; &nbsp; &nbsp; &nbsp;a. A lysate<br />&nbsp; &nbsp; &nbsp; &nbsp;b. A plaque picked into buffer<br />&nbsp; &nbsp; &nbsp; &nbsp;c. An environmental sample<br />&nbsp; &nbsp; &nbsp;d. A sample from an experiment (e.g., a one-step)<br />5. A 35 &deg;C water bath

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

[DESCRIPTION]
This protocol describes purification of OPTN-GST.

[STEPS]
SECTION: Purification of OPTN-GST
1. To purify OPTN-GST, Clone human OPTN cDNA in a pETDuet-1 vector with an C-terminal GST-tag.
SECTION: Purification of OPTN-GST
2. After the transformation of the pETDuet-1 vector encoding OPTN-GST in E. coli Rosetta pLysS cells, grow the cells in 2xTY medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C.

SECTION: Purification of OPTN-GST
3. Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 960 min at 18 °C
SECTION: Purification of OPTN-GST
4. Collect the cells by centrifugation and resuspend in lysis buffer.

Lysis buffer 
 
 
SECTION: Purification of OPTN-GST
5. Sonicate cell lysates.
SECTION: Purification of OPTN-GST
6. Clear the lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
SECTION: Purification of OPTN-GST
7. Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind OPTN-GST.
SECTION: Purification of OPTN-GST
8. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of OPTN-GST
9. Wash beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash buffer 
 
 
High-salt wash buffer 
 
 
SECTION: Purification of OPTN-GST
10. Elute the proteins 120 min with 20 millimolar (mM) reduced L-glutathione in 50 millimolar (mM) HEPES  pH 7.4, 300 millimolar (mM) NaCl, 1 millimolar (mM) DTT buffer.
SECTION: Purification of OPTN-GST
11. Collect the supernatant, filter through a 0.45 µm syringe filter, concentrate using a 50 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).
SECTION: Purification of OPTN-GST
12. Elute the proteins with SEC buffer.

SEC buffer 
 
 
SECTION: Purification of OPTN-GST
13. Analyze  fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification of OPTN-GST
14. Pool fractions containing purified OPTN-GST.
SECTION: Purification of OPTN-GST
15. After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at -80 °C.
SECTION: Purification of OPTN-GST
5.1. Sonicate cell lysates twice for 30 s (1/2).
SECTION: Purification of OPTN-GST
5.2. Sonicate cell lysates twice for 30 s (2/2).
TITLE: PBMC Isolation from apheresis collars
AUTHORS: Girija Goyal, Girija Goyal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Commonly used protocol to isolate peripheral blood mononuclear cells from whole human blood modified here for apheresis collars</div></div>

[STEPS]
?. Acquire collar and make an incision to drain blood into 50mL conical tube.
?. Dilute blood product with 2X volume RPMI or PBS. Mix well.
?. Slowly layer solution on top of 10 mL density gradient solution.
?. Centrifuge at 300 g for 25 minutes at room temperature. Set acceleration and deceleration levels to minimal.
22 °C
?. Remove white layer of PBMCs using a 5 mL pipette tip.
?. Add these cells to 10 mL warm media in a 50 mL tube.
?. If using 5 ml or more of the Leukopak, you may have a very high number of cells. To effectively wash them, fill tube to 50 mL.
?. Centrifuge at 120 g for 10 minutes to remove platelets and get an accurate count. Return acceleration / deceleration levels to high or 9.
?. Aspirate media and resuspend cells in 20 mL warm  media per 10 ml of starting blood product. Steps 10-12 can be optimized depending on your yield.
?. Dilute 100x by adding 10ul cell solutions to 890 ul media in a 1ml eppendorf tube and 100ul Trypan blue
?. Count cells using a hemocytometer. Count the number of cells in each of the four quadrants. Use the following formula to find the total number of cells.total # of cells = (cells counted /4)X dilution factorX 104 cells/ml
?. Cells can be kept in solution in the refrigerator for up to two hours.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Indirect immunofluorescence infectivity assay for reovirus using LICOR Imaging System</p>

[BEFORE_START]
<p>Plates and reagents:</p>
<p>Corning Black with clear bottom TC treated 96 well plates (Corning #3904)</p>
<p>DRAQ5 #4084 from Cell Signaling</p>
<p>Sapphire 700 #928-40022 from LICOR</p>
<p>Antibody 800 from LICOR (Life Technologies ones don’t work for this)</p>

[GUIDELINES]
<p>This assay is standardized to for 96-well plates using reovirus polyclonal antiserum. New batches of polyclonal antiserum or monoclonal antibodies should be tested to determine optimal dilution.</p>

[STEPS]
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TITLE: DNA extraction (BOMB)
AUTHORS: Tsu-Chun  Hung, Yin-Tse    Huang

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 1mm beads to 2ml enppendorf tube

SECTION: Sample Collection
2. Add200 µL of 0.5mm beads to 2ml enppendorf tube

SECTION: Sample Collection
3. Add 225 µL of TE buffer to 2ml enppendorf tube
 

SECTION: Sample Collection
4. Add 375 µL of lysis buffer to 2ml enppendorf tube
 


SECTION: Sample Collection
6. Collect 10-20 mg of sample to 2ml enppendorf tube
SECTION: Sample crush
7. Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 4 min 
SECTION: Centrifugation
8. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: DNA purification
9. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
9.1. Add 125 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 

 


SECTION: DNA purification
9.2. Add 200 µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate 
 

SECTION: DNA purification
10. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate 
SECTION: DNA purification
11. Add 300 µL of 80% enthanol to the  3rd well of 96 deep well plate 
SECTION: DNA purification
12. Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate 
SECTION: DNA purification
13. Add 100 µL of DD water to the 5th well of 96 deep well plate 
SECTION: DNA purification
14. Put the prepared 96 deep well plate in the automated DNA extraction machine

SECTION: Sample Collection
5. Add 267 µL of 10M ammonium acetate to 2ml enppendorf tube
 

SECTION: DNA purification
15. After the extraction is done, collect 100 µL of the eluted sample as the DNA template for downstream experiments
TITLE: Working Alone in the Lab 
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Working alone in the laboratory should be avoided; however, trained personnel can work alone in the laboratory if they adhere to the following Standard Operating Procedure (SOP). Students in training (during at least the 1st semester of laboratory work) cannot work alone in the laboratory.</div></div>

[STEPS]
TITLE: Passaging and plating A549 cells
AUTHORS: Jcprice 

[STEPS]
?. Remove and discard culture medium, by tipping dish carefully to one side and aspirating the media without disturbing the adherent cells
?. Briefly rinse the cell layer with 1 mL 0.05% (w/v) Trypsin - 53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
?. Add 0.5mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5  to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
?. Add 3 to 4 mL of complete growth medium and aspirate cells by gently pipetting.
?. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C for at least an hour.  Cells will adhere but not grow well until EDTA/trypsin is asperated off.
?. After cells have adhered to plate carefully aspirate away the media and feed cells new media
?. Allow cells to grow at 37°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The DNA amount can be measured for each chromosome of the karyotype by image cytometry. Image cytometry (ICM) associates microscopy, digital image and software technologies and has been particularly useful in spatial and densitometric cytological analyses.  ICM integrates the methodologies of microscopy optical calibration, standard density filters, digital CCD camera and image analysis softwares for quantitative applications. Besides of all system calibration and setup, cytological protocols must perform good slide preparations for efficient and reliable ICM analysis. </p>

[GUIDELINES]
<p>Silva JC, Carvalho CR, Clarindo WR (2018) Updating the maize karyotype by chromosome DNA sizing. PLoS ONE 13(1): e0190428. https://doi.org/10.1371/journal.pone.0190428</p>
<p> </p>
<p>Abreu IS, Carvalho CR, Clarindo WR (2008) Chromosomal DNA content of sweet pepper determined by association of cytogenetic and cytometric tools. Plant Cell Reports 27(7): 1227–1233. https://doi.org/10.1007/s00299-008-0539-4</p>
<p> </p>
<p>Carvalho CR, Clarindo WR, Abreu IS (2011) Image cytometry: nuclear and chromosomal DNA quantification. In: Chiarini-Garcia H, Melo RCN (Eds) Light microscopy: methods and protocols. Humana Press, New York, USA, 51–68. https://doi.org/10.1007/978-1-60761-950-5_4</p>
<p> </p>
<p>Chieco P, Jonker A, Van Noorden CJF (2001) <em>Image cytometry: Microscopy handbooks 46. </em>Springer-Verlag, New York, 128 pp.</p>
<p> </p>
<p>Doležel J, Bartoš J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99-110</p>
<p> </p>
<p>Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA and genome size of trout and human. Cytometry 51:127-128</p>
<p> </p>
<p>Greilhuber J (2005) Intraspecific variation in genome size in Angiosperms: identifying its existence. Ann Bot 95:91-98</p>
<p> </p>
<p>Greilhuber J, Ebert I (1994) Genome size variation in <em>Pisum sativum</em>. Genome 37:646-655</p>
<p> </p>
<p>Hardie DC, Gregory TR, Hebert PDN (2002) From pixels to picograms: a beginners’ guide to genome quantification by Feulgen image analysis densitometry. J Histochem Cytochem 50:735-749</p>
<p> </p>
<p>Vilhar B, Dermastia M (2002) Standardization of instrumentation in plant DNA image cytometry. Acta Bot Croat 61:11-26</p>
<p> </p>
<p>Vilhar B, Greilhuber J, Koce JD, Temsch EM, Dermastia M (2001) Plant genome size measurement with DNA image cytometry. Ann Bot 87:719-728</p>
<p> </p>
<p> </p>

[STEPS]
TITLE: Bright Field Image Capture with Zeiss AxioImager
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the system that is used to capture Z stack images which are reassembled post-processing into 3D reconstructions.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the procedure for performing a co-assembly of short reads to obtain contigs using the Megahit assembler. This procedure is performed on a node at the UoA HPC due to memory considerations. </p>

[GUIDELINES]
<p><a href="http://rc.arizona.edu/hpc-htc/using-systems" target="_blank">UoA HPC: Using the Systems</a></p>
<p> </p>
<p><a href="https://github.com/voutcn/megahit" target="_blank">Megahit github</a></p>

[STEPS]
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TITLE: QSAR Prediction
AUTHORS: BOC  Sciences

[DESCRIPTION]
The early linear free-energy approaches developed by Hansch and Free-Wilson have provided a fundamental scientific framework for the quantitative correlation of chemical structure with biological activity and spurred many developments in the field of quantitative structure-activity relationships (QSAR). QSAR prediction methods are attempting to predict the toxicological endpoint and breadth of mechanisms which are complicated. The quality and quantity of the available biological toxicology data could be another obstacle in the modeling process.


[STEPS]
TITLE: Fungi Permanent Storage
AUTHORS: You Li, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to store and revive ambrosia fungi.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a variation of the CagA translocation assay performed in the lab. </p>
<p>The protocol was used in the publication DOI: <a href="https://doi.org/10.1111/cmi.12166" target="_blank">10.1111/cmi.12166</a></p>

[STEPS]
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TITLE: Cloning gene of interest into attb plasmid for PhiC31 integration
AUTHORS: Goran  Tomic

[DESCRIPTION]
This protocol describes a simple cloning procedure to insert a gene of interest (GOI) into an attb plasmid to use in combination with PhiC31 integration to generate low-copy integrants. 


[STEPS]
1. The vector is generated in-house from the pX28 vector from Addgene (#46850).  Not available from a repository yet, so synthesise/clone your own based on the sequence provided with this protocol.
2. Linearise the attb vector with MfeI and XhoI (purify the backbone). Alternatively , PCR-amplify the backbone using high-fidelity DNA polymerase. I use PrimeSTAR from Takara with excellent results (https://www.takarabio.com/products/pcr/high-fidelity-pcr/primestar-hs-dna-polymerase)

If worried about introducing any mutations by PCR, sequence the entire plasmid using Plasmidsaurus service.
3. Design primers to amplify the GOI to insert into attb plasmid. Use Takara primer design tool to generate primers that overlap with the ends of the vector backbone to achieve seamless cloning using In-Fusion cloning kit (https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools). Alternatively, use your preferred method for cloning (ligation, Gibson assembly, etc.)


4. In-Fusion cloning (if using this method)

3 μL of DNA mix (insert and vector, 200 ng in total) 
1 μL of In-Fusion enzyme mix 
1 μL dH2O
Total volume 5 μL

15 min at 50C
5. Transformation protocol:


Stellar Competent Cells (Use 14 mL round-bottom tubes) 
50 uL of cell suspension + 2.5 uL of DNA mix Incubate on ice for 30 min
Heat shock for 45 s at 42 degrees, keep on ice 1-2 min 
Add 450 uL SOC medium (warmed up to 37 degrees previously)
Incubate for 1 h at 37 degrees (30 min is also fine if in a rush) 
Plate 50 uL on an Amp agar plate Leave in the incubator at 37C

Alternatively, use any other competent cells and transformation protocol of choice.
TITLE: Visualization of RNA using gel electrophoresis method. 
AUTHORS: khawla alshuraiqi

[DESCRIPTION]
For the analysis of RNA samples, polyacrylamide gel electrophoresis (PAGE) is a highly effective technique. Information about the sample composition and the structural integrity of each unique RNA species is provided by denaturing PAGE. Moreover, Various techniques can be used to see RNA in gels, depending on the type of detection reagent used. Numerous dyes, such as ethidium bromide, SYBR green, and toluidine blue, can be used to stain RNA molecules. 

[STEPS]
1. For sample preparation, all samples should be at the same initial concentration by diluting samples with nuclease-free water with a total volume of 9ul and 1 ul for loading dye.
3. Running buffer preparation (we can reuse it) 
A.    From the stock of 50 X prepare 1X by adding 7 ml in 343 ml deionized water.
2. Agarose gel preparation 
A.    Into a flask add 1 ml TAE 50X buffer     
B.    49 ml deionized water. 
C.     0.75 g agarose powder to make 1.5 % agarose. 
D.    Heat in the microwave for 1 min, and shake heat again for 1 min. 
E.     Add the fluorescence dye (Midori Green) 
F.     Put cast in the sealer. 
G.    Insert combs according to the number of samples. 
H.    Let it dry (usually takes 10 min)  
    
4. Running the gel 
1.     Take the gel with its cast and put it in the middle of the tray. 
2.     Pour the running puffer until it is higher than the wells by 2 mm. 
3.     Load DNA ladder
4.     Mix 9 ul of RNA sample with 1 ul of loading dye using parafilm. 
5.     Load samples carefully.
 6.    Cover the gel tray, make sure black to black and red-to-red connections. 
7.     Connect to the power supply, and run it for 40 min, 80 V. 
8.     Keep your eyes on the gel to ensure perfect separation. 
9.     Take the gel to the documentation system. 
10.  Chose Nucleic Acid gel settings, and capture. 
11.  There should be 2 clear bands one at 28 (thick band) Twice intense as the other band, and the other at 18 (thinner band).