Amplification Grade DNase I

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.

Features and Benefits

Convenient

Eliminates DNA from RNA preparations

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Amplification Grade DNase I

Product Number	Product Description	Quantity
AMP-D1	Amplification Grade DNase I (Sufficient for treating at least 1,000 µg of RNA)	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

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

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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