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[DESCRIPTION]
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<p>Invitrogen’s patented Syto dyes are cell-permeant cyanine dyes that bind to nucleic acids. Several Syto dyes are available with varying cell permeability, fluorescence enhancement upon binding to nucleic acids, excitation and emission spectra, and nucleic acid selectivity and binding affinity. The Syto 60 stain has absorption and fluorescence emission maxima of 652/678 nm. Nucleic acids stained with the Syto 60 stain can be detected and quantified on the Odyssey® Infrared and Odyssey Fc Imaging Systems using the 700 nm channel.</p>
<p> </p>
<p>In the procedures outlined, the Syto 60 dye was used to stain serial dilutions of a 1 kb DNA ladder and a 50 bp DNA ladder (New England Biolabs, P/N N3232 and N3236, respectively). Three methods are presented for staining of DNA in this technical note. The Syto 60 stain can be included in the DNA sample for detection using an Odyssey Imaging system.The Syto 60 stain can also be combined with ethidium bromide (EtBr) and included in the DNA sample for visualization on an Odyssey Imaging System and on a UV transilluminator; or the Syto 60 stain can be diluted and used alone as a post-electrophoresis gel stain. For additional information, see Imaging Nucleic Acid Gels on the Odyssey Fc Imager.</p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
<p> </p>
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<p>Invitrogen’s patented Syto dyes are cell-permeant cyanine dyes that bind to nucleic acids. Several Syto dyes are available with varying cell permeability, fluorescence enhancement upon binding to nucleic acids, excitation and emission spectra, and nucleic acid selectivity and binding affinity. The Syto 60 stain has absorption and fluorescence emission maxima of 652/678 nm. Nucleic acids stained with the Syto 60 stain can be detected and quantified on the Odyssey® Infrared and Odyssey Fc Imaging Systems using the 700 nm channel.</p>
<p> </p>
<p>In the procedures outlined, the Syto 60 dye was used to stain serial dilutions of a 1 kb DNA ladder and a 50 bp DNA ladder (New England Biolabs, P/N N3232 and N3236, respectively). Three methods are presented for staining of DNA in this technical note. The Syto 60 stain can be included in the DNA sample for detection using an Odyssey Imaging system.The Syto 60 stain can also be combined with ethidium bromide (EtBr) and included in the DNA sample for visualization on an Odyssey Imaging System and on a UV transilluminator; or the Syto 60 stain can be diluted and used alone as a post-electrophoresis gel stain. For additional information, see Imaging Nucleic Acid Gels on the Odyssey Fc Imager.</p>
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<p><strong>II. Methods</strong></p>
<p><strong> </strong></p>
<p><strong>Method I. Electrophoretic Staining</strong></p>
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<p>Purpose: To obtain an archivable, digital image of a DNA agarose gel using an Odyssey Imaging System.</p>
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<p><em>NOTE: This method may not be optimal for visualizing bands smaller than 100 bp.</em></p>
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<p>See 'STEPS' for method. </p>
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<p><strong>Method II. Dual Electrophoretic Staining</strong></p>
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<div class="column">Purpose: To obtain a digital image using an Odyssey® Imaging System and then visualize DNA bands on a UV transilluminator for excision.<br /> <em>NOTE: This method may not be optimal for visualizing bands smaller than 100 bp.</em></div>
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<div class="column">See 'STEPS' for method. </div>
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<p>Odyssey or Odyssey CLx Imaging System Settings:</p>
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<p>Gel face down on scan bed</p>
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<p>700 nm channel intensity: 5-8 • Focus offset: 0.5 mm</p>
<p>Odyssey Sa or Aerius System Settings:</p>
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<p>Gel face down on scan bed</p>
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<p>700 nm channel intensity: 5-8</p>
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<p>Focus offset: 1.7 - 2.0 mm</p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hgrc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hgqc2w6.png" data-ofn="Screen Shot 2016-12-29 at 9.44.55 AM.png" /></p>
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<p>Odyssey Fc System Settings:</p>
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<p>Gel face up on imaging tray</p>
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<p>Acquisition time: 2 min.</p>
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<p>UV Transilluminator:</p>
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<li>Place gel on UV transilluminator to identify bands for excision. If the band(s) to be excised are not bright enough, the gel can be soaked for a short time in a 2 mg/mL solution of EtBr inTAE orTBE buffer after imaging on an Odyssey System.</li>
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<p><strong>Hints and Tips for Methods I and II</strong></p>
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<p>The range of dilution for the Syto 60 stain is 1:500 to 1:20,000. The dilution to use is dependent on the DNA size, concentration, and whether the Syto 60 stain will be used in combination with EtBr.<br />NOTE: This method may not be optimal for visualizing bands smaller than 100 bp.</p>
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<p>The Syto 60 stain, diluted within the recommended range in TE buffer, is stable for 1 week at 4 °C.</p>
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<p>EtBr is not stable in TE and should be diluted fresh each time.</p>
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<p>The grade of agarose is important. High grade or Molecular Biology grade agarose is less likely to cause “speckling” on Odyssey images.</p>
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<p>When using the Odyssey, Odyssey CLx, Odyssey Sa, and Aerius to image DNA gels stained with Syto 60 stain, it may be necessary to scan the gel with the front side on the glass and/or adjust the focus offset, depending on the gel thickness. A 5 mm - 7 mm thick gel is optimum.</p>
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<p>Addition of EtBr to the gel and running buffer with the Syto 60 stain added in the sample is not recommended.</p>
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<div class="column"><strong>Method III. Post-electrophoretic staining</strong></div>
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<p>Purpose: To obtain an archivable, digital image of a DNA agarose gel using an Odyssey Imaging System.<br />NOTE: This method IS recommended for visualizing &lt;100 bp.</p>
<p> </p>
<p>See 'STEPS' for method. </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hgsc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hgsc2w6.png" data-ofn="Screen Shot 2016-12-29 at 9.47.39 AM.png" /></p>
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<p><strong>Recommended Dilutions and Time Requirements for Method III.</strong></p>
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<p>Gel Conditions: A 10 x 10 cm agarose gel, 5-8 mm thick, made with high-grade or molecular biology-grade agarose in 1X TAE or TBE buffer</p>
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<p>The quickest staining time was 5 minutes using 1:2000 dilution of the Syto 60 stain in water. Gels were stained sufficiently in 15 minutes using a 1:2500 dilution. A 1:5000 dilution of Syto 60 stain requires at least 30-45 minutes of staining. The most dilute solution tested was 1:20,000 and the gel was stained sufficiently after 45 minutes. There was no significant improvement in sensitivity from 60 to 120 minutes using 1:10,000, 1:15,000 and 1:20,000 dilutions.</p>
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<table style="height: 228px;" width="223">
<tbody>
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<td style="width: 97.203125px;">
<p><strong>Syto 60</strong></p>
<p><strong>Nucleic Acid</strong></p>
<p><strong>Stain</strong></p>
<p><strong>Dilution</strong></p>
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<td style="width: 109.8125px;">
<p><strong>Minimum</strong></p>
<p><strong>Staining Time</strong></p>
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<td style="width: 97.203125px;">1:2000</td>
<td style="width: 109.8125px;">5-15 min</td>
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<td style="width: 97.203125px;">1:2500</td>
<td style="width: 109.8125px;">15-30 min</td>
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<td style="width: 97.203125px;">1:5000</td>
<td style="width: 109.8125px;">30-45 min</td>
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<td style="width: 97.203125px;">1:10000</td>
<td style="width: 109.8125px;">45 min</td>
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<td style="width: 97.203125px;">1:15000</td>
<td style="width: 109.8125px;">45 min</td>
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<td style="width: 97.203125px;">1:20000</td>
<td style="width: 109.8125px;">45 min</td>
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<p><strong>Speckle Reduction</strong></p>
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<p>The appearance of speckles on the gel may be present after<br />post-electrophoretic staining. Use the Odyssey Application software’s 'FILTER” then “Noise Removal' function, or Image Studio’s “NOISE REDUCTION” function, to improve the appearance of the images (see Figure 4).To reduce the appearance of speckles on the gel, cut off the wells before post-electrophoretic staining and rinse the gel in water.</p>
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<p><em> NOTE: The type and concentration of agarose will affect the degree of speckling. For example, low melting-point agarose tends to be highly prone to speckling.</em></p>
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<p><em> </em></p>
<p><em><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hgtc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hgtc2w6.png" data-ofn="Screen Shot 2016-12-29 at 9.51.26 AM.png" /></em></p>
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<p><strong>III. Conclusions</strong></p>
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<p>A table of cost comparisons for the Syto 60 stain and the ethidium bromide staining reagents used for each method is provided below. The recommended dilution of the Syto 60 staining reagent makes it more competitive with ethidium bromide on a cost basis, and the small amount of Syto 60 stain used in the sample is environmentally friendly.</p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hguc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hguc2w6.png" data-ofn="Screen Shot 2016-12-29 at 9.52.30 AM.png" /></p>
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[STEPS]
SECTION: Method I. Electrophoretic Staining
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SECTION: Method II. Dual Electrophoretic Staining
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SECTION: Method III. Post-electrophoretic staining
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SECTION: Method III. Post-electrophoretic staining
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SECTION: Method III. Post-electrophoretic staining
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