SYBR Gold

Advantages: More sensitive than EtBr Allows post-stain processing of DNA, e.g., restriction digest or Southern blotting Replaces silver staining and radioactive labeling in some applications, e.g., SSCP Can be used with glyoxal and formaldehyde gels Quick quantitative analysis using the Totallab software SYBR gold can be used in the presence of urea, glyoxal and formaldehyde No need to destain because of low background Allows excision of bands for cloning after heteroduplexing Excitation Maximum: 300 nm Emission Maximum: 537 nm

Post-Staining: Run gel

Use centrifuge to spin stock solution to bottom of tube.
Dilute SYBR Gold to a final dilution of 1:10,000 in TE, TBE, or TAE (pH 7.0-8.5).
Leave gel to shake in dye solution for 10-30 minutes, longer for thicker or higher percentage gels.
No destaining step necessary.

Pre-Staining: Incubate DNA with 1:5,000 SYBR gold for 15 minutes, prior to electrophoresis.
Add loading dye after 15 minute incubation.

Pre-casting: (Preferred method for high percentage gels)
Dilute SYBR Gold to 1:10,000 into gel solution just prior to casting.
Add 1:10,000 dilution of SYBR Gold to running buffer.

Binding: SYBR Gold is thought to bind non-covalently to the phosphate backbone of the DNA molecule.

Detection Limits: Detection of ssDNA and RNA is less sensitive than for dsDNA.
Exact figures for ssDNA are not currently available.
Sensitivity will always be better in acrylamide gels than in agarose gels because acrylamide gels have lower background.
Post-staining: in acrylamide: ~100 pg dsDNA/band depending on band size, 1-3 ng ssDNA or
RNA, 10-20 ng synthetic 24-mer oligonucleotide.
~400 pg in 0.8% agarose

Pre-staining: ~150 pg in 6% acrylamide, ~450 pg in 0.8% agarose

Linear Range: ~1.5-2 orders of magnitude.

Emission Filters: SYBR gold filter

Useful tips for SYBR Gold

Use double-gloved protection against DMSO stock solution.
Cover dye solution with aluminum foil to prevent bleaching of fluorophore by overhead lighting.
SYBR Gold and EtBr may compete for DNA binding.
Staining first with EtBr, then with SYBR Gold will give better results than with EtBr alone, but
inferior to pure SYBR Gold staining.
Avoid use of bind silane with SYBR Gold on polyacrylamide gels; it is preferable to
use a Gel-Slick-type product on the opposite plate.
Staining of extra large sandwich gels can be easily accomplished by laying the gel
sandwich onto paper towels, having cleaned the glass plates well first.
Remove top glass plate then pipette enough 1:10,000 SYBR Gold stain onto gel to cover

completely. Spread evenly with Pasteur pipette and leave protected from light for 10-60 minutes.

If required, SYBR Gold can be removed from dsDNA by simple ethanol precipitation.
Bring solution up to 10Mm NaCl, add 2.5 volumes 95% ethanol, incubate 20 minutes on ice, centrifuge for 10 min at 4C.

Storage: In DMSO in plastic, protected from light. Stock solution kept at -20 degrees C is stable for six to twelve months. Diluted reagent kept at 4 degrees C is stable for three to four days.

Handling: Treat as potential mutagen, as data is unavailable.

Vendor: Molecular Probes, Inc. www.probes.com