Lucigen/BaselineZERO™ DNase/DB0715K/5,000 MBU

Applications

• Removal of genomic DNA from small-sample total RNA preparations for expression analysis (Fig. 1)
• Removal of small DNA oligonucleotides (e.g., random primers)

Baseline-ZERO™ DNase* digests dsDNA and ssDNA into mononucleotides more effectively than the commonly used bovine pancreatic DNase I. Even the small DNA oligonucleotides that remain after treatment with bovine pancreatic DNase I are undetectable by gel electrophoresis following treatment with Baseline-ZERO DNase (Fig. 2). Removal of DNA from RNA preparations is particularly beneficial when RNA in a sample is amplified using a method that involves reverse transcription using random primers, since any contaminating DNA would also be a template for random-primed cDNA synthesis.

Figure 1. Real-time PCR of HeLa RNA preparations treated with various DNases. The lower the C_T value (intersection of curves with the red line), the greater the amount of residual DNA not digested by the indicated DNase. Thus, Baseline-ZERO™ DNase removed all detectable DNA from the RNA sample. The TaqMan® probe assay amplified a 268-bp fragment of β-actin. Samples were run in duplicate.

Unit Definition: One Molecular Biology Unit (MBU) of Baseline-ZERO™ DNase produces an increase in the A₂₆₀ of a solution of dsDNA, of 0.001 per minute at 25°C. Functionally, 1 MBU completely digests 1 µg of linear pUC19 DNA to mononucleotides in 10 minutes at 37°C.

Storage Buffer: Baseline-ZERO DNase is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 10 mM CaCl₂, 10 mM MgCl₂ and 0.1% Triton® X-100.

10X Baseline-ZERO™ DNase Reaction Buffer: 100 mM Tris HCl (pH 7.5), 25 mM MgCl₂ and 5 mM CaCl₂.

10X Baseline-ZERO™ DNase Stop Solution: 30 mM EDTA.

Quality Control: Baseline-ZERO DNase is assayed for its ability to completely digest linear dsDNA to mononucleotides under standard assay conditions. Baseline-ZERO DNase is free of detectable RNase activities as assayed by PAGE analysis of 1 µg of a synthetic RNA transcript following an overnight incubation with sufficient DNase I to completely digest 1,000 µg of DNA.

References

1. Kienzle, N. et al. (1996) BioTechniques 20, 612.

*Patent pending.