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[DESCRIPTION]
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<p>The Odyssey Fc Imager, with 600 channel capabilities, can image agarose gels stained with popular DNA stains, such as ethidium bromide and SYBR Safe DNA stain, with sub-nanogram sensitivity. The Odyssey Fc Imager contains a 532 nm diffuse source with an excitation maximum of 520 nm and a detection maximum of 600 nm. These instrument parameters are within the range of the excitation and emission wavelengths of ethidium bromide (Ex/Em = 302 &amp; 518/605 nm) and other visible fluorescent nucleic acid stains and provide a sensitive gel documentation option.</p>
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<p>SYBR Safe DNA stain (Ex/Em = 502/530 nm) has also been tested on the Odyssey Fc Imager (using the 600 channel) with sensitivities exceeding ethidium bromide detection. The maximum fluorescence emission wavelength of SYBR Safe is very close to the maximum excitation wavelength. However, the Odyssey Fc 600 channel collects excitation light at a wavelength 50 nm higher than the maximum excitation wavelength of SYBR Safe. These instrument properties decrease the background and improve the signal-to-noise ratio for nucleic acid detection.</p>
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<p>Specific instructions are given in this protocols for ethidium bromide and SYBR Safe use. Other nucleic acid binding stains may also be compatible with the Odyssey Fc Imager. Please check the excitation and emission spectra of each stain.</p>
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<p>Developed for: Odyssey Fc Imaging System</p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
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<p>The Odyssey Fc Imager, with 600 channel capabilities, can image agarose gels stained with popular DNA stains, such as ethidium bromide and SYBR Safe DNA stain, with sub-nanogram sensitivity. The Odyssey Fc Imager contains a 532 nm diffuse source with an excitation maximum of 520 nm and a detection maximum of 600 nm. These instrument parameters are within the range of the excitation and emission wavelengths of ethidium bromide (Ex/Em = 302 &amp; 518/605 nm<sup>1,2</sup>) and other visible fluorescent nucleic acid stains and provide a sensitive gel documentation option.</p>
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<p>SYBR Safe DNA stain (Ex/Em = 502/530 nm) has also been tested on the Odyssey Fc Imager (using the 600 channel) with sensitivities exceeding ethidium bromide detection. The maximum fluorescence emission wavelength of SYBR Safe is very close to the maximum excitation wavelength. However, the Odyssey Fc 600 channel collects excitation light at a wavelength 50 nm higher than the maximum excitation wavelength of SYBR Safe. These instrument properties decrease the background and improve the signal-to-noise ratio for nucleic acid detection.</p>
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<p>Specific instructions are given in this technical note for ethidium bromide and SYBR Safe use. There are a variety of commercial DNA stains that may be appropriate for fluorescent imaging with the Odyssey Fc 600 channel. SYBR Green I (Life Technologies), GelStar (FMC), Gel RedTM (Biotium), Gel GreenTM (Biotium) and Nancy-520 (Sigma) stains have also been tested at LI-COR. Other nucleic acid binding stains may also be compatible with the Odyssey Fc Imager. Please check the excitation and emission spectra of each stain.</p>
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<p>The Odyssey Fc Imager is also equipped with two infrared channels (700 and 800) and a chemiluminescent detection channel. Nucleic acid detection in the 700 channel is achieved with Syto® 60 stain, a cell-permeant cyanine dye. A detailed protocol is available for the use of Syto 60 with the Odyssey and Aerius family of imagers (LI-COR, Syto 60 Staining of Nucleic Acids in Gels).</p>
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<p><em><strong>Note: </strong>Any questions regarding specific properties of the DNA binding stains should be directed to the representative vendors listed in this technical guide.</em></p>
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<p><strong>II. DNA Separation and Detection on Agarose Gels</strong></p>
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<p><strong>A. Suggested Materials</strong></p>
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<p>This section is intended as a guideline; other materials may be substituted.</p>
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<p>High Grade or Molecular Biology Grade agarose</p>
<p>(Low melting-point agarose may increase the degree of speckling on the digital image.) <strong>OR</strong></p>
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<p>E-Gel® Pre-cast agarose gels from Life Technologies (Ethidium Bromide, SYBR® Safe, or Clear gel</p>
<p>types)</p>
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<p>1X TAE or TBE buffer</p>
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<p>Ethidium Bromide (EtBr, 10 mg/mL solution) <strong>OR</strong></p>
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<p>SYBR Safe DNA stain (10,000X concentrate in DMSO)</p>
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<p>Gel tank and casting tray for running submersion gels</p>
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<p>Power supply</p>
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<em><strong>Note:</strong> Dispose of all gel and buffer solutions in accordance with the regulations of your facility.</em></div>
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<div class="column"><strong>B. In-Gel Pre-Staining Protocol</strong></div>
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<div class="column">See 'STEPS'</div>
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<div class="column"><strong>C. Post-Electrophoresis Staining Protocol</strong></div>
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<div class="column">See 'STEPS'</div>
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<div class="column"><strong>D. E-Gel Pre-Cast Agarose Gels</strong></div>
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<p>The E-Gel pre-cast agarose gels containing Ethidium Bromide or SYBR Safe are compatible with digital imaging on the Odyssey Fc Imager using the 600 channel. The clear versions of the E-Gel gels allow for post-staining with a DNA binding stain of your choice. Follow the manufacturer’s protocols for sample preparation and gel electrophoresis parameters.</p>
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<p><strong>IV. Results - Ethidium Bromide and SYBR Safe</strong></p>
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<p><strong>A. Sensitivity of the Odyssey Fc Imager, 600 Channel</strong></p>
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<p>The images in Figure 1 were prepared following the post-electrophoresis staining protocol on page 4 with Ethidium Bromide and SYBR® Safe DNA stains. These images show the sensitivity of the Odyssey Fc Imager.</p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/herc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/herc2w6.png" data-ofn="Screen Shot 2016-12-21 at 10.41.02 AM.png" /></p>
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<p><strong>B. DNA Samples - Plasmid Digests and PCR PRoducts</strong></p>
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<p>DNA samples were loaded on 1.2% E-Gel® gels (Ethidium Bromide and SYBR Safe), electrophoresed for 30 minutes, and imaged on the Odyssey Fc Imager (2 minutes) in E-Gel cassettes.</p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hesc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hesc2w6.png" data-ofn="Screen Shot 2016-12-21 at 10.42.03 AM.png" /></p>
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<p><strong>C. Examples of Other DNA Stains</strong></p>
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<p>The same DNA samples from Figure 2 were loaded on 1.2% agarose gels pre-stained with the DNA stains as specified. Images were acquired on the Odyssey Fc Imager using the 600 channel.</p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/hetc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/hetc2w6.png" data-ofn="Screen Shot 2016-12-21 at 10.42.53 AM.png" /></p>
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<p><strong>V. References</strong></p>
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<p>1. Waring MJ., (1965) J Mol Biol. 13(1):269-82 Complex formation between ethidium bromide and nucleic acids</p>
<p>2. LePecq JB, Paoletti C., (1967) J Mol Biol. 27(1):87-106 A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization</p>
<p>LI-COR Biosciences, (2010) Syto 60 Staining of Nucleic Acids in Gels<br /> LI-COR Biosciences, (2011) How to Adjust the Lookup Tables in Image Studio for an Optimal Image Display</p>
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[STEPS]
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