Description
RNase-free DNase I
DNase I, RNase-free (1 U/μl) is an RNase-free DNase I for removing genomic and contaminating DNA from RNA preparations and enzymatic reactions. It efficiently digests single- and double-stranded DNA and is easily inactivated before downstream steps. It is supplied at 1 U/μl with a 10× Reaction Buffer.
DNase I is an endonuclease that cleaves phosphodiester bonds to produce mononucleotides and single- or double-stranded oligonucleotides with 5'-phosphorylated and 3'-hydroxylated ends. Its activity depends on Ca2+ and is also activated by Mg2+ and Mn2+. With Mg2+, it cleaves dsDNA nonspecifically at any site on either strand. With Mn2+, it cleaves both strands at almost the same sites, giving blunt ends or sticky ends with 1–2 nt overhangs. The recombinant enzyme is purified from non-animal hosts. It is used for RNA extraction, in vitro transcription, DNA removal in RT-PCR, DNase I footprinting, nick translation and DNA library preparation. For research use only.
Key Features
| RNase-free | Removes DNA while keeping RNA preparations intact for downstream use. | |
| Digests ssDNA and dsDNA | Endonuclease activity degrades both single- and double-stranded DNA. | |
| Easy inactivation | Inactivate with 5 mM EDTA at 65°C for 10 min or by phenol/chloroform extraction. | |
| Animal-free source | Recombinant enzyme purified from non-animal hosts. |
Specifications
| Enzyme | Recombinant DNase I, RNase-free |
| Concentration | 1 U/μl |
| Source | Recombinant, purified from non-animal hosts |
| Unit definition | One unit completely degrades 1 μg of pUC19 plasmid DNA at 37°C in 10 min |
| Cofactors | Ca2+-dependent; activated by Mg2+ and Mn2+ |
| Applications | RNA extraction, in vitro transcription, DNA removal in RT-PCR, DNase I footprinting, nick translation, DNA library preparation |
| Shelf life | 2 years |
Components
| Components | 1,000 U | 10,000 U |
| DNase I, RNase-free (1 U/μl) | 1 ml | 10 ml |
| 10× Reaction Buffer | 1 ml | 10 ml |
Storage
Store at -30 ~ -15°C.
Removing DNA from RNA before RT-PCR
- Set up the digestion
- Prepare the following mixture in an RNase-free tube:
| Components | Volume |
| RNase-free ddH2O | To 10 μl |
| 10× Reaction Buffer | 1 μl |
| DNase I, RNase-free (1 U/μl) | 1 μl |
| RNA | X |
- Mix gently by pipetting and incubate at 37°C for 15 min.
- Stop the reaction
- Add 1 μl EDTA (50 mM) to the 10 μl reaction, mix gently by pipetting and incubate at 65°C for 10 min.
- Proceed to RT-PCR
- Use the treated RNA directly as template for RT-PCR.
Removing template DNA after in vitro transcription
- Add DNase I
- Add 1 U of DNase I per 0.5 μg of template DNA to the transcription reaction.
- Digest
- Mix gently by pipetting and incubate at 37°C for 15 min.
- Inactivate
- Inactivate DNase I by phenol/chloroform extraction.
Q1: How do I inactivate DNase I after digestion?
- Add EDTA to a final concentration of 5 mM and heat at 65°C for 10 min, or remove the enzyme by phenol/chloroform extraction.
Q2: What inhibits DNase I activity?
- Chelating agents, Zn2+ at certain concentrations, 0.1% SDS, reducing agents such as DTT and mercaptoethanol, and salt concentrations above 50–100 mM.
Q3: How much enzyme should I use to remove template DNA after in vitro transcription?
- Add 1 U of DNase I for every 0.5 μg of template DNA and incubate at 37°C for 15 min, then inactivate by phenol/chloroform extraction.
Q4: How is the unit defined?
- One unit is the amount of enzyme that completely degrades 1 μg of pUC19 plasmid DNA at 37°C in 10 min.
