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Description: Amplification Grade DNase I (Deoxyribonuclease I) is an endonuclease isolated from bovine pancreas that digests double- and single-stranded DNA into oligo- and mono-nucleotides. The DNase I is suitable for eliminating DNA from RNA preparations prior to sensitive applications, such as RT-PCR. Since no RNA purification procedure removes 100% of the DNA, RNA samples should be digested with DNase I before RT-PCR. A simple 15 minute digestion at room temperature removes the contaminating DNA. The DNase I is inactivated by adding the stop solution provided and heating. Heating also denatures the RNA, so the RNA can be used directly for reverse transcription.
Many commercial DNase I formulations are contaminated with residual RNases. This RNase contamination can destroy or degrade valuable RNA samples prior to reverse transcription. Laboratory comparisons have shown that Sigma's Amplification Grade DNase I demonstrates lower RNase activity than that from several leading molecular biology product suppliers.
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Features and Benefits
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Convenient
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Eliminates DNA from RNA preparations |
Amplification Grade DNase I
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Product Number
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Product Description
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Quantity
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Amplification Grade DNase I (Sufficient for treating at least
1,000 µg of RNA)
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1 Kit
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Kit Contents
- Amplification Grade DNase I (1,000 units)
- 10X reaction buffer
- Stop Solution
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Sigma's Amplification Grade DNase I has the lowest RNase activity
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Figure 1. A 1 µg aliquot of a 1.9 kb in vitro transcription product was incubated with 1 unit of each DNase I at 37°C for 1 hour and analyzed on a 1% agarose gel. Cu = unincubated control (RNA in buffer without DNase, kept on ice).
Ci = incubated control (RNA in buffer but without DNase, incubated at 37°C for 1 hour).
Note: To determine the effectiveness of DNase I treatment, parallel PCR reaction should be run without adding reverse transcriptase to check for amplification from contaminating DNA.
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RT-PCR sensitivity with or without DNase digest
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Figure 2. RNA was prepared from HeLa cells with the GenElute Mammalian Total RNA Kit (RTN). A 1000, 100, 10, 1 or 0.1 ng aliquot of RNA was digested with Amplification Grade DNase I and amplified by RT-PCR. * Indicates reactions without reverse transcriptase.
Note: Without DNase treatment, a PCR product is obtained without reverse transcriptase, indicating that the RNA is contaminated with genomic DNA. DNase treatment eliminated this PCR-only product. RT-PCR products are visible down to 0.1 ng RNA with or without DNase, demonstrating no loss of sensitivity with the DNase treatment. |
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