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Nucleic Acid Stains

SYBR® stained gels can be visualized with a standard UV transilluminator, a laser-based scanner equipped with an excitation source in the UV range or between 470 and 530 nm, or a blue-light transilluminator.

You can add ethidium bromide to the sample loading dye or to the RNA samples to a final concentration of 10 µg/mL for direct visualization of the RNA during and after electrophoresis. Adding ethidium bromide to the samples or loading buffer is preferable to adding it to the gel or poststaining the gel, since formaldehyde in the gel will interact with the ethidium bromide, resulting in fluorescence, making it difficult to discern specific staining. Using 10 µg/mL ethidium bromide reportedly reduces assay sensitivity by 5–10%. However, the benefit of knowing that the RNA is intact, that the gel ran well, and that all of the RNA transferred, can be worth this minor loss in sensitivity.

Yes, you can stain your TBE gels with ethidium bromide, SYBR® Green I, SYBR® Green II, and the SilverXpress® Silver Staining Kit. For ethidium bromide staining, soak the gel in a 2 µg/mL solution of ethidium bromide in ultrapure water for 20 minutes. Destain by rinsing with three successive 10-minute rinses of ultrapure water. Visualize bands under UV light.

Ethidium bromide can be used to detect ssDNA, RNA, and dsDNA. This fluorescent dye intercalates between the stacked bases of nucleic acids, and exhibits an increased fluorescence at 590 nm. Ethidium bromide can detect down to ~1–10 ng/band of dsDNA in an agarose gel.

For use in agarose gels:

  • Add ethidium bromide to melted agarose to a final concentration of 0.5 µg/mL. Do not melt agarose already containing ethidium bromide.

For staining agarose gels after electrophoresis:

  • You can stain gels that have been run in the absence of ethidium bromide by covering the gel in 0.5 µg/mL ethidium bromide in water and gently agitating for 10 to 30 minutes.
  • If necessary, gels can be destained by shaking in H2O for an additional 30 minutes. To stain RNA gels, you should minimize staining time and definitely include a destaining period.

SYBR® Green I is used for staining dsDNA and ssDNA. SYBR® Green II is used for RNA. These stains are preferred if you will use gel scanning devices to image the gel. In most applications, SYBR® Gold is a bit more sensitive than either SYBR® Green I or SYBR® Green II (because it is a better general-purpose stain), and it is preferred for documentation with black and white photography. SYBR® Safe DNA Gel Stain is a reduced mutagenicity formula designed for use with blue light systems. It is somewhat less sensitive than the other forms.

The binding mode of the SYTO® nucleic acid stains is unknown. However, the behavior of these and related nucleic acid dyes suggests the following binding properties:

  1. They appear to contact the solvent (suggested by sensitivity to salt, divalent cations, and in particular, SDS) and thus are likely to have contacts in the grooves.
  2. All SYTO® dyes appear to show some base selectivity and are thus likely to have minor groove contacts.
  3. They can be removed from nucleic acid via ethanol precipitation; this characteristic is not shared by ethidium bromide and other intercalators. Likewise, the dyes are not removed from nucleic acid via butanol or chloroform extraction. These extraction methods do remove ethidium bromide from nucleic acid.
  4. SYTO® binding is not affected by nonionic detergents.
  5. SYTO® dyes are not quenched by BrdU, so they do not bind nucleic acids in precisely the same way as Hoechst 33342 and DAPI ((4′,6-diamidino-2-phenylindole).
  6. SYBR® Green I has shown little mutagenicity on frameshift indicator strains, indicating that it isn't likely to strongly intercalate.

Disposal regulations vary. Please contact your safety office or local municipality for disposal guidelines.

SYBR® Safe Stain is non-mutagenic and non-toxic. It shows very similar sensitivity in the UV range, ~500 pg/band, with equivalent resolution to ethidium bromide. For imaging, ethidium bromide is visualized using a standard UV transilluminator, while SYBR® Safe™ stain can be viewed with UV (~280 nm), laser scanners with visible light capability, or blue light (500 nm).

All SYBR® dyes have similar spectral properties, but have different chemical compositions. SYBR® Safe™ DNA Gel Stain was specifically developed as a safer alternative to ethidium bromide. SYBR® Green I is an ultrasensitive stain for dsDNA, and SYBR® Green II is a highly sensitive stain for RNA and ssDNA. All SYBR® dyes are optimally excited by the Safe Imager™ Blue-Light Transilluminator.

Yes. Simply substitute a SYBR® Safe™ DNA Gel Stain solution for the buffer when preparing the molten agarose. If you are using the 10,000X SYBR® Safe™ DNA Gel Stain concentrate, dilute the concentrated stain 1:10,000 in agarose gel buffer (e.g., 1X TBE or 1X TAE) and add the buffer/stain solution to the powdered agarose. You can heat the agarose/SYBR® Safe™ DNA Gel Stain mixture briefly in the microwave.

The SYBR® Safe™ DNA Gel Stain content in a 1X solution is less than 1 ppm.

DNA stained with SYBR® Safe™ DNA Gel Stain can be viewed using a blue-light transilluminator such as our Safe Imager™ 2.0 Blue-Light Transilluminator or a standard UV transilluminator. If you plan to use the DNA for cloning, avoid exposing DNA stained with SYBR® Safe™ DNA Gel Stain to UV light.

Not really. Most deep amber/orange ethidium bromide filters have a cutoff value around 550 nm. The SYBR® Green dyes emit at 520 nm, so you are filtering out your data by using this filter.

Unlike UV light, blue light causes minimal damage to DNA and is therefore safer for you and better for your DNA sample. Use of SYBR® Safe DNA Gel Stain and the Safe Imager™ Blue-Light Transilluminator gives improved cloning efficiency over DNA stained with ethidium bromide and exposed to UV light.

Bands stained with SYBR® Safe DNA Gel Stain are visible to the eye on a 300 nm transilluminator. However, you can obtain optimum detection by photographing the gel using a UV-compatible emission filter with your CCD or film camera. UV bulbs may also emit some infrared (IR); if your camera lens is not specially coated to block IR, an IR-blocking filter is needed to prevent the appearance of faint images of the UV bulbs behind your gel. For optimum detection and complete safety, use the Safe Imager™ Blue-Light Transilluminator.

Please go here and click on the “Filter Recommendations” tab to see filter recommendations for use with SYBR® Safe™ DNA Gel Stain. Note that the excitation and emission spectra of SYBR® Safe™ DNA Gel Stain are very similar to those of SYBR® Green I, SYBR® Green II, and SYBR® Gold dyes, as well as fluorescein (FITC). Therefore, filters appropriate for these dyes can also be used. A camera filter is not required with the Safe Imager™ Blue-Light Transilluminator; the amber filter provided with the instrument serves this purpose.

Some ethidium bromide filters allow the transmission of all light above 500 nm. These filters (which are often yellow) and their associated camera settings can be used with SYBR® Safe™ DNA Gel Stain, usually with only minor adjustments to the exposure or gain. Other ethidium bromide filters (often red) only transmit light around or above 600 nm; these filters and their associated camera settings are not suitable for use with SYBR® Safe™ DNA Gel Stain.

SYBR® Safe™ DNA Gel Stain has two main excitation peaks: in the UV region at 280 nm, and in the visible region at 502 nm. Thus, 254 nm or 300 nm UV excitation will work, as will excitation with 488 nm lasers, 470 nm LEDs, and broad blue light sources (such as the Safe Imager™ Blue-Light Transilluminator (Cat. No. S37102). Maximal excitation occurs at 502 nm; the Safe Imager™ Blue-Light Transilluminatorr is therefore the best choice for excitation of SYBR® Safe™ DNA Gel Stain. You can find the full excitation and emission spectra for SYBR® Safe™ DNA Gel Stain online and also in the protocol provided with the stain.

SYBR® Safe™ DNA Gel Stain is compatible with all downstream applications we have tested so far, including excising PCR products from gels, gel purification, Gateway® cloning, TOPO® cloning, and restriction enzyme cloning. If you have a unique application that works with SYBR® Safe™ DNA Gel Stain, send us the details at techsupport@lifetech.com.

It can vary depending on your specific gels, imaging system, and sample/standard type.
The data below are for UV excitation. For 488 nm excitation, we had a 12 pg sensitivity limit for both SYBR® Green I and SYBR® Gold dyes on the Bio-Rad Molecular Imager FX™ system, which has a 488 laser.

Standard

Detection limit

Wavelength

Stain

M13 (7.5 kb ssDNA)

100 pg/band
300 pg/band
1 ng/band

254 nm
300 nm
300 nm

SYBR® Green I
SYBR® Green I
Ethidum bromide

23S and 16S rRNA

100 pg/band
1 ng/band
3–400 pg/band

254 nm
254 nm
300 nm

SYBR® Green I
Ethidium bromide
SYBR® Green I or II*

24-mer oligo

1 ng/band

254 nm

SYBR® Green I

* SYBR® Green II performed slightly better than SYBR® Green I
10% polyacrylamide gel:


24-mer oligo

10 ng/band
200 ng/band
2 ng/band

254 nm
254 nm
254 nm

SYBR® Green II
Ethidium bromide
SYBR® Green I

5 % urea polyarylamide gel:


24-mer oligo

10–30 ng/band
1–200 ng/band

254 nm
254 nm

SYBR® Green II
Ethidium bromide

We strongly discourage the reuse of SYBR® Safe™ DNA Gel Stain, as this practice significantly lowers sensitivity.

SYBR® Safe™ DNA Gel Stain is easily removed from nucleic acids by ethanol precipitation.

Similarly to ethidium bromide, SYBR® Safe™ DNA Gel Stain runs in the opposite direction of the migrating DNA. This has no practical effect on the use of gels cast with SYBR® Safe™ DNA Gel Stain, as only the very bottom of the gel will have a lower concentration of stain. This effect can be partially counteracted by adding SYBR® Safe™ DNA Gel Stain to the running buffer.

The SYBR® dyes are useful only over a narrow range of pH, from about 7 to 8. Outside this range, the fluorescent signal diminishes rapidly.

Nucleic Acid Ladders and Markers

TrackIt™ DNA Ladders contain two tracking dyes in the sample buffers, which serve as visual markers for tracking electrophoresis progress through the gel, and also to indicate when maximum resolution is achieved. The tracking dyes should not obscure your visualization of DNA bands in the ladder, as the dyes run outside the limits of most DNA bands in the ladder. TrackIt™ Cyan/Orange Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Orange G. We recommend TrackIt™ Cyan/Orange Loading Buffer for DNA fragments between 10 bp and 1 kb. The TrackIt™ Cyan/Yellow Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Tartrazine. We recommend TrackIt™ Cyan/Yellow Loading Buffer for DNA fragments between 100 bp and 10 kb. The molecular weights are Xylene Cyanol FF, 638.6; Orange G, 452.4; Tartrazine, 534.4. Note: The TrackIt™ DNA Ladders are not recommended for use with polyacrylamide gels and are not designed for quantitation.

We offer three different buffers: BlueJuice™ Gel Loading Buffer, TrackIt™ Loading Buffers, and E-Gel® Sample Loading Buffer. BlueJuice™ Gel Loading Buffer can be used for agarose and native polyacrylamide gels, including E-Gel™ Agarose Gels. The buffer is supplied at 10X, and should be used at 2X for agarose gels or 1X for polyacrylamide gels. TrackIt™ Loading Buffers are supplied at a 6X concentration, and contain tracking dyes as visualization markers for migration. We offer two versions, the TrackIt™ Cyan/Orange Loading Buffer containing Xylene Cyanol FF and Orange G dyes and the TrackIt™ Cyan/Yellow Loading Buffer containing Xylene Cyanol FF and Tartrazine dyes. The E-Gel® Sample Loading Buffer is supplied at a ready-to-use 1X concentration for specific use with E-Gel® and E-Gel® EX Agarose Gels. The buffer contains Xylene Cyanol FF and Tartrazine dyes. For more information, please use this link. Do not heat your ladder sample before loading. Ladders can alternatively be diluted in TE containing a final concentration of 20 mM NaCl if running on agarose gels. Apply approximately 0.1 µg of ladder per mm lane width.

1 Kb Plus DNA Ladder

1 Kb DNA Ladder

Low molecular weight bands in 50 and 100 bp increments

Low molecular weight bands in variable size increments

The 1 kb repeats are exactly 1 kb

The 1 kb repeats are exactly 1,018 bp repeats

Low molecular weight bands are mostly the same intensity as 1 kb bands

There is a gap between the 506 and 1,018 bp band

All bands have the same overhang (GATC)

Some bands are blunt and some bands have an AvaI overhang

Orientation band at 1,650 bp

Orientation band at 1,636 bp

20 bands

18 bands

Our Low DNA Mass Ladder (Cat. No. 10068013) and High DNA Mass Ladder (Cat. No. 10496016) were specifically created for quantitative estimation of DNA mass in gels.

Yes, we offer our Ambion® Millenium™ Markers and Ambion® RNA Ladders for single-stranded RNA. Please use this link to view our RNA Marker Selection Guide.