Nucleic Acid Dye
- Diamond™ Nucleic Acid Dye (Cat.# H1181)
- SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO; Life Technologies, Cat.# S-11494)
- SYBR® Safe DNA Gel Stain (Life Technologies, Cat.# S-33102)
- Agarose, Low Melting Point, Analytical Grade (Cat.# V2111)
- Molecular Imager® Gel Doc™ XR System (Bio-Rad, Cat.# 170-7950EDU )
Dilution of Nucleic Acids
DNA: Four-fold serial dilutions of BenchTop 1kb DNA Ladder were prepared in 1X Blue/Orange Loading Dye
RNA: Two-fold serial dilutions of RNA Markers were prepared in TE Buffer.
Gel Staining Method
For all DNA sample, 10µl (100ng/µl) was run per lane in 0.8% agarose gels. Gels containing DNA samples were run for 45 minutes at 100 volts. For all RNA samples, 3µl of RNA was added to 3µl of formaldehyde loading dye. RNA samples were incubated for 5 minutes at 70°C and then run on a 1% agarose gel for 60 minutes at 75 volts.
All stains are provided as 10,000X stock concentrations in DMSO. After electrophoresis, all gels were stained in a 1:10,000 dilution of due for 30 minutes, protected from light.
DNA and RNA samples were prepared as previously described. Concentrated stains were diluted 1:10,000 in molten agarose (0.8% for DNA and 1% for RNA) prior to casting. Gels were run, protected from light, for 45 minutes at 100 volts for DNA samples and 60 minutes at 100 volts for RNA samples.
Preloading Method (DNA Only)
DNA samples were prepared as previously described. Dyes were diluted in 1X Blue/Orange Loading Dye to a final dilution of 1:10,000 for DNA samples. Gels were run protected from light for 45 minutes at 100 volts.
Visualization of Nucleic Acids
All gels were imaged on the Molecular Imager® Gel Doc™ XR System using the SYBR® Gold or the SYBR® Safe setting.