Application Guide Imaging Nucleic Acid Gels on Odyssey® Imagers Published July 2021. The most recent version of this document is posted at licor.com/bio/support. Page 2 - Imaging Nucleic Acid Gels on Odyssey® Imagers Table of Contents Page I. Background 2 II. Introduction 3 III. Stain Comparison 4 IV. Separation and Detection Protocol 5 Suggested Materials 5 In-Gel Prestaining 6 Post-Electrophoresis Staining 7 E-Gel® Pre-Cast Agarose Gels 8 V. Imaging Protocol 8 I. Background A common method for separating and analyzing nucleic acid molecules is nucleic acid gel electrophoresis. Nucleic acid gel electrophoresis separates nucleic acids based on molecular size and subsequent ability to move through an agarose gel. Depending on the experiment, a nucleic acid sample can be stained during or after separation. The gel is placed in the electrophoresis apparatus and buffer added to submerge the gel. Nucleic acid samples and size markers are loaded then separated on the gel using an electric field. The stained and separated nucleic acid molecules are visualized in the gel then compared to the band markers to estimate their sizes. There are numerous commercial stains that may be appropriate for imaging the results of nucleic acid electrophoresis. Common stains include Ethidium Bromide (EtBr), SYBR® Safe DNA stain, SYTO™ 60 Nucleic Acid Stain, and GelRed® Nucleic Acid Stain. Note: Always verify that a stain’s excitation and emission wavelengths are compatible with the intended instrument prior to use. Imaging Nucleic Acid Gels on Odyssey® Imagers - Page 3 II. Introduction This technical note explains the respective stain imaging capabilities of each Odyssey® Imager and the D-DiGit® Gel Scanner and how to effectively detect and image results. Each Odyssey Imager and the D-DiGit Gel Scanner images specific stains. Depending on the desired stain, the choice of imaging system can vary. Note: Any inquiries regarding specific properties of a given nucleic acid stain should be directed to the respective vendor. Table 1. Excitation and emission of common commercial stains used with the Odyssey M Imager. EtBr SYBR SYTO GelRed Pro-Q™ GelGreen MIDORI GreenView Safe 60 Nucleic Diamond ® Nucleic Green ™ DNA Gel DNA Nucleic Acid Phosphoprote Acid Gel Advanc Stain Gel Acid Stain in Gel Stain Stain e Stain Stain Nucleic Acid Stain Excitatio 520 488 685 520 520 500 290/490 500 n (nm) Emission 570- 519- 721-740 570-610 570-610 530-550 530 530 (nm) 610 543 Table 2. Excitation and emission of common commercial stains used with the Odyssey XF Imager and Odyssey Fc Imager. EtBr SYBR Safe GreenView DNA DNA Gel Gel Stain Stain Excitation (nm) 520 520 685 Emission (nm) 573-637 573-637 716-746 Table 3. Excitation and emission of common commercial stains used with the Odyssey DLx Imager and Odyssey CLx Imager. EtBr Excitation (nm) 685 Emission (nm) 710-730 The D-DiGit Gel Scanner is designed to scan gels stained with safe alternatives to EtBr. The D-DiGit Gel Scanner is intended for use with the following stains. Page 4 - Imaging Nucleic Acid Gels on Odyssey® Imagers l SYBR Safe DNA Gel Stain l SYBR Gold Nucleic Acid Gel Stain l SYBR Green Nucleic Acid Gel Stain l GelRed Nucleic Acid Gel Stain l Diamond Nucleic Acid Dye l GelGreen Nucleic Acid Gel Stain l Midori Green Direct l Midori Green Advance DNA Stain l GreenView Nucleic Acid Gel Stain l GreenView Plus Nucleic Acid Gel Stain l GreenView Ultra DNA Gel Stain Note: Some stains may be more sensitive than others, allowing for visualization of smaller amounts of nucleic acid. For more information on the D-DiGit® Gel Scanner, see the D-DiGit Gel Scanner User Guide. III. Stain Comparison The following figure compares the results of three representative stains (i.e., SYBR Safe Stain, GelRed Stain, and EtBr) that were imaged on the Odyssey® M Imager. Imaging Nucleic Acid Gels on Odyssey® Imagers - Page 5 Figure 1. Dilutions (250 to 2 ng) of a 1 kb ladder were loaded on a 1% agarose gel. Gels were post-stained with A) 1:10,000 dilution of SYBR® Safe in 1X TAE buffer for 40 minutes, B) 3X GelRed stain in H2O for 30-45 minutes, and C) Ethidium Bromide for 30-45 minutes. Images were collected on the Odyssey® M Imager in the A) 530 nm and B, C) 590 nm channels. IV. Separation and Detection Protocol The following protocol explains how to perform DNA separation and detection on agarose gels using nucleic acid gel stain. Suggested Materials This section describes the suggested materials for DNA separation and detection on agarose gels. It is intended as a guide; other materials may be substituted. l High Grade or Molecular Biology Grade agarose OR E-Gel® Pre-cast agarose gels (Ethidium Bromide or SYBR Safe) (Life Technologies) Note: A low melting-point agarose may increase the degree of speckling on the digital image and is more difficult to use. l 1X TAE or TBE buffer l Ethidium Bromide (EtBr, 10 mg/mL solution) OR SYBER Safe DNA Stain (10,000X concentrate in DMSO) OR GelRed Nucleic Acid Stain (10,000X concentrate in DMSO) l Gel tank and casting tray for running submersion gels l Power supply Page 6 - Imaging Nucleic Acid Gels on Odyssey® Imagers Note: Dispose of all gel and buffer solutions in accordance with the regulations of your facility. In-Gel Prestaining This section describes the steps for in-gel prestaining. Note: Prestaining may cause higher background than post-staining techniques. Commonly used stains: EtBr and SYBR Safe DNA Gel Stain Gel Preparation 1. Prepare desired agarose (0.8%, 1.0%, 1.2%, etc.) in 1X TAE or 1X TBE buffer. 2. Heat and dissolve the agarose in buffer using a microwave oven. 3. Cool solution until warm to the touch (60 °F) prior to adding a DNA stain. Ethidium Bromide: Stock solutions are typically 10 mg/mL. Add EtBr to give a final concentration of 0.5 µg/mL. SYBR Safe: Stock solutions are typically 10,000X. Add SYBR Safe to a final concentration of 1X. 4. Pour molten agarose solution into casting tray and set comb into place. Allow gel to solidify. 5. Remove comb and place gel in buffer tank. Do not allow the gel to become dry. Note: Both EtBr and SYBR Safe are positively charged stains and will migrate in the opposite direction of the DNA. If the stain is included only in the gel but not the buffer, there will be an area of high background indicating the stain has not migrated out of the gel. Buffer Preparation 1. Prepare enough buffer (1X TAE or 1X TBE) to fill the apparatus and cover the gel. 2. Add a DNA stain to buffer. Ethidium Bromide: Add 5 µL of 10 mg/mL EtBr stock solution to 100 mL of buffer (final concentration: 0.5 µg/mL). Imaging Nucleic Acid Gels on Odyssey® Imagers - Page 7 SYBR Safe: Add 10 µL of 10,000X SYBR Safe stock solution to 100 mL of buffer (final concentration: 1X). Electrophoresis and Destaining 1. Prepare samples with loading buffer and load in gel. 2. Electrophorese samples at 5-8 V/cm. 3. (Optional) Destain in water for 15-20 minutes. Repeat as necessary to remove background from the gel. 4. Image on an Odyssey® Imager in the appropriate channel. Post-Electrophoresis Staining This section describes the protocol for post-electrophoresis gel staining. Commonly used stains: EtBr and SYBR Safe DNA Gel Stain Gel Preparation 1. Prepare desired agarose (0.8%, 1.0%, 1.2%, etc.) in 1X TAE or 1X TBE buffer. 2. Heat and dissolve the agarose in buffer using a microwave oven. 3. Cool solution until warm to the touch (60 °F) prior to pouring in casting tray. 4. Pour molten agarose solution into casting tray and set comb into place. Allow gel to solidify. 5. Remove comb and place gel in buffer tank. Do not allow the gel to become dry. Buffer Preparation 1. Prepare enough buffer (1X TAE or 1X TBE) to fill the apparatus. Electrophoresis 1. Prepare samples with loading buffer and load in gel. 2. Electrophorese samples at 5-8 V/cm. 3. When complete, remove gel from apparatus and place in a container suitable for staining, (i.e., black incubation box). Add enough stain to completely cover gel. Stain for approximately one hour with gentle agitation. Refer to the manufacturer's recommendation for stain concentration. 4. Rinse the stained gel in water for 15 minutes and image. If desired, the gel may be rinsed again to try to decrease any remaining background fluorescence. Page 8 - Imaging Nucleic Acid Gels on Odyssey® Imagers E-Gel® Pre-Cast Agarose Gels The E-Gel pre-cast agarose gels containing EtBr or SYBR Safe are compatible with digital imaging on the D-DiGit® Gel Scanner or the Odyssey® XF Imager or Odyssey Fc Imager using the 600 channel. Follow the manufacturer’s protocols for sample preparation and gel electrophoresis parameters. Note: E-Gel gels must be imaged through the plastic cover. V. Imaging Protocol The following protocol explains how to image stained nucleic acids on agarose gels using Odyssey Imagers or the D-DiGit Gel Scanner. Each imager and scanner’s protocol and imaging software varies slightly. Please reference the imager or scanner’s respective steps in Table 4 when preparing to image. DNA bands can be excised from a gel directly on the plexiglass casting tray of the D-DiGit Gel Scanner. For detailed steps on band excision, please see the D-DiGit Gel Scanner User Guide. Table 4. Protocols for imaging stained nucleic acids with LI-COR imaging systems.