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Invitrogen™ DNase I, Amplification Grade

Catalog No. 18068015
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100 U
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18068015 100 U
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Catalog No. 18068015 Supplier Invitrogen™ Supplier No. 18068015
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Description

Includes

DNase I, Amp Grade (100U), 10X DNase I Reaction Buffer, 25mM EDTA (pH 8.0)

Purified and tested for nondetectable levels of RNase contamination

DNase I, Amplification Grade, digests single- and double-stranded DNA to oligodexyribonuleotides containing a 5' phosphate. DNase I, Amplification Grade, is suitable for eliminating DNA during critical RNA purification procedures such as those prior to RNA-PCR amplification. Absence of RNase is tested by performing a ribonuclease assay with RNA ladder.

  • Specific activity is >10,000 units/mg
  • Source: Purified from bovine pancreas
  • Performance and quality testing: Ribonuclease assay with RNA ladder and ability to digest single-stranded and double-stranded DNA to oligonucleotides are determined
  • Unit definition: One unit increases absorbance of high molecular weight DNA solution at rate of 0.001 A260 units/min./mL of reaction mixture at 25°C
  • Unit reaction conditions: 0.1M sodium acetate (pH 5.0), 5mM MgCl2, 50 μg/mL calf thymus DNA, and enzyme in 1mL for 10 min. at 25°C

Removing DNA from RNA and protein preparations, PCR & Real-Time PCR, Real Time PCR (qPCR), Reverse Transcription

Order Info

Shipping Conditions: Approved for shipment on wet or dry ice

Specifications

Content And Storage Contents:
1 vial of DNase I, Amp Grade (100 U)
1 vial of 10X DNase I Reaction Buffer (1000 μL)
1 vial of 25 mM EDTA (pH 8.0) (200 μL)

Store at -20°C in a non-frost-free freezer.
Shipping Condition Approved for shipment on Wet or Dry Ice
Enzyme DNase
Compatible Buffer Reaction Buffer
Quantity 100 U

Frequently Asked Questions (FAQs)

How can RNA be treated to remove residual DNA?

RNAse-free DNase treatment of the RNA can reduce DNA to undetectable levels. We recommend using our DNase 1, Amplification Grade (Cat. No. 18068015).

Does Thermo Fisher Scientific offer a protease-free DNase?

We do not test for protease activity as part of our QC but there is PMSF (a protease inhibitor) in the storage buffer. Furthermore, in the preparation of DNase I, we use a soybean trypsin inhibitor column to remove proteases.
How should I treat my RNA sample prior to RT-PCR to ensure that I have no DNA contamination?

DNase I treatment is optional, and one has to consider individual experimental design.

Potential disadvantage of omitting the DNase I step: you may get amplification from genomic DNA. If you omit this step, you will need to include a no RT control and design primers that will not amplify genomic DNA, like those spanning two different exons or exon-exon junctions.

Potential benefit of omitting the DNAse I Step:
saves time; consumes less reagent, saves pipetting steps, and reduces RNA loss (important for precious samples).

Protocol for DNAse I treatment:
Combine 1 µg total RNA, 1 µL 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 20 mM MgCl2), 1 µL Amplification Grade DNAse I (1 unit/µL), and DEPC-treated water to 10 µL. Incubate for 15 min at room temperature. Inactivate by adding 1 µL of 25 mM EDTA and heat for 10 min at 65 degrees C.
Note: 1 U of DNAse I should be enough to treat up to ~10 µg of RNA.

To locate the manual for Amplification Grade DNAse I, search www.thermofisher.com with the Cat. No.18068-015. The manual will be one of the links on the product page.
What is the difference between DNase I and Amplification Grade DNase I?

The Amplification Grade DNase I (Cat. No. 18068-015) is subjected to an extra final HPLC purification step to remove traces of RNases. The Amplification Grade DNase I is supplied as 1 unit/µL and comes with 10X buffer (200 mM Tris-HCL pH 8.4, 20 mM MgCl2, 500 mM KCl) and a vial of 25mM EDTA.

In RT-PCR, a large excess of Amplification Grade DNase I could be used to digest an RNA template without degradation of the RNA (in-house data). Use Amplification Grade DNase I to remove genomic DNA carryover in RNA samples prior to RT-PCR.

The regular DNase I is supplied at 5-15 mg/mL (50-375 U/µL) and does not come with its own buffer.
How can I inactivate DNase I?

Add of 1 µL of 25 mM EDTA solution to the reaction mixture in 10 µL reaction with 1 unit DNase I, Amplification Grade (or 1:1 molar ratio of Mg++ ions:EDTA) to chelate the Mg++ ions in the DNase I buffer. Heat for 10 min at 65 degrees C.

Note: It is vital that the EDTA be at least at 2 mM prior to heat-inactivation to avoid Mg-dependent RNA hydrolysis.

DNA-free and Turbo-free versions of DNase I can be inactivated with included DNase Inactivation Reagent.
What is the specific activity of your DNase I?

Specific activities vary by lot. Our DNase I must have a specific activity greater than 10,000 units/mg to pass our quality standards. Average specific activities vary between 10,000-25,000 units/mg.
Are your DNase I products RNase-free?

Most of our DNase I products are guaranteed free of RNase activity. However, please note that Cat. No. 18047-019 is not tested for RNAse and is recommended primarily for protein applications. The other products are suitable for removing DNA from both RNA and protein preparations, for nick translating DNA, and for generating random fragments of DNA. For more demanding RT-PCR applications, we recommend using DNAse I, Amplification Grade.
How can I eliminate DNA contamination from my RNA prior to RT-PCR?

We recommend using Amplification-Grade DNase I, Cat. No. 18068-015, or an equivalent product to eliminate DNA contamination. Combine 1 µg total RNA, 1 µL 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 20 mM MgCl2), 1 µL DNAse I, Amplification Grade, 1 unit/µL, and DEPC-treated water to 10 µL. Incubate for 15 min at room temperature. Inactivate by adding 1 µL of 25 mM EDTA and heat for 10 min at 65 degrees C. Note: 1 unit of DNAse I should be enough to treat up to ~10 µg of RNA. The detailed protocol can be found in the product manual. Simply search the Catalog Number on our website to find a copy on the product detail page.

For Research Use Only. Not for use in diagnostic procedures.