Nucleic Acid Gel Imaging

After electrophoretic separation of nucleic acid samples, you are ready to stain your DNA gels, image, and analyze. With LI-COR, you have imaging options.

Use the D-DiGit® Gel Scanner to image and extract DNA bands without concern for UV damage to your DNA samples or the hazards related to UV light and ethidium bromide. You can scan nucleic acid gels (large or small; image area: 17.8 cm x 12.7 cm), excise bands, document your work and analyze your data with this compact, easy-to- use gel scanner. After capture, images can be imported into Image Studio™ Lite Software for convenient analysis, annotation and storage.

See the complete list of safe stains compatible with the D-DiGit Scanner.

Use the 700nm channel of Odyssey® CLx Imager (image area: 25 cm x 25 cm), Odyssey Sa Imager, or Odyssey Fc Imager (Figure 1) to image DNA gels stained with SYTO® 60 fluorescent nucleic acid stain.

For more information, read the detailed protocol entitled Imaging Nucleic Acids Gels on the Odyssey Fc Imager. You can also refer to SYTO 60 Staining of Nucleic Acids in Gels for protocols on how to use near-infrared fluorescent dyes to stain nucleic acids and image on Odyssey Imaging Systems.

figure 1
Figure 1. DNA gels imaged on the Odyssey Fc using Ethidium Bromide, SYBR® Safe and SYTO 60. The ethidium bromide gel was also documented using Polaroid film to show the comparison.
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Figure 2. DNA Gel imaged on the D-DiGit DNA Gel Scanner using SYBR Safe. A 1:2 dilution series of Lambda DNA – BstEII Digest was run on 0.8% agarose gels containing 1X TAE buffer. Lane 1 contains 250 ng of DNA; sample loading volumes decreased progressively down to 2 ng in Lane 8. The gel was stained post-electrophoresis with 1X SYBR Safe (Invitrogen) and imaged on the D-DiGit Gel Scanner using a 6X exposure setting.

Ethidium Bromide (EtBr) Imaging on the Odyssey Fc Imager

Sensitivity is high and background fluorescence is low when using SYTO 60 red fluorescent nucleic acid stain to visualize DNA gels on the LI-COR Odyssey Infrared Imagers. The detection sensitivity and lower limit of detection for SYTO 60 with any of the Odyssey Imagers are better than those of ethidium bromide detected with either a Polaroid camera or a CCD imaging system.

If you need to image ethidium bromide, SYBR Safe DNA stains, or other DNA stains in your nucleic acid gels in the 600nm region, use the Odyssey Fc Imaging System.

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Figure 3. Comparison of detection limits between ethidium bromide. A DNA gel stained with ethidium bromide was imaged with a Polaroid or CCD camera and detection limits were compared to a DNA gel stained with SYTO 60 imaged on the Odyssey Infrared Imager.

Table 1. SYTO 60: Limit of Detection (LOD) with Odyssey Imaging

DNA Fragment (bp) LOD (pg) LOD (fmol)
100 375 5.7
500 95 0.29
1500 44 0.044

Table 2. Ethidium Bromide: Limit of Detection (LOD) with Polaroid Imaging

DNA Fragment (bp) LOD (pg) LOD (fmol)
100 3000 45
500 1500 4.5
1500 700 0.7

Table 3. Ethidium Bromide: Limit of Detection (LOD) with CCD Imaging

DNA Fragment (bp) LOD (pg) LOD (fmol)
100 750 11.4
500 375 1.14
1500 175 0.18

DNA Gel Staining Cost Comparisons

SYTO dyes are cell-permeant cyanine dyes that bind to nucleic acids. Nucleic acids stained with the SYTO 60 stain can be detected and quantified on the Odyssey CLx, Odyssey Classic, Odyssey Sa, and Odyssey Fc Imaging Systems using the 700nm channel.

This application note, "SYTO 60 Staining of Nucleic Acids in Gels" (2010), presents 3 methods for using the SYTO 60 stain. The choice of method depends on the result the user desires.

  • Use Method I to obtain an archivable, digital image of a DNA agarose gel using an Odyssey Imaging System.
  • Use Method II if digital image with an Odyssey Imaging Systems is required prior to visualizing DNA bands on a UV transilluminator for excision of target bands.
  • Use Method III as a post-staining method if the time to obtain the DNA image is not restricted since this method requires at least 45 minutes of post-electrophoresis staining and also uses more concentrated stain.
Stain Dilution Staining Method Cost
SYTO 60 stain 1:1000 Method I or II $0.006 (1μI/well, 8 wells)
SYTO 60 stain 1:20000 Method I $0.0003 (1μI/well, 8 wells)
SYTO 60 stain 1:2500 Method III $7.44 (25 ml)
EtBr 1:500 Method II $0.00007 (1μI/well, 8 wells)
EtBr 1:2000 Method III $0.05 (25 ml)

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Figure 4. Comparison of DNA in Agarose Gels Stained with SYTO60 and Ethidium Bromide and Imaged with the Odyssey Classic, the Odyssey Fc and a UV Transilluminator to Acquire a Polaroid Film Image. A 1.2% agarose gel was imaged using the Odyssey Imaging System (panel A), Odyssey Fc Imaging System (panel B) or a UV transilluminator and the image captured using Polaroid 667 film (Panel C). Lane 1) 1μg 1Kb ladder Lane 2) 0.5 μg 1Kb ladder Lane 3) 0.25 μg 1Kb ladder Lane 4) 0.5 μg pUC 19 Lane 5) 0.5 μg pUC19/HindIII / XmnI Lane 6) 1 μg 50bp ladder Lane 7) 0.5 μg 50bp ladder Lane 8) 0.25 μg 50bp ladder. The gel was electrophoresed for 8 V/cm in 1X TAE buffer for 1 hr. The Odyssey intensity setting for the 700nm channel was 8 and focus offset was 0.5 with the gel face down. The Odyssey Fc acquisition was 2 minutes, gel face up.

Resources

Technical Notes

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