Description
Concentrated RNase-free DNase I
DNase I, RNase-free (50 U/μl) removes 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. This concentrated 50 U/μl format comes with a dilution buffer and 10X reaction buffer.
DNase I is an endonuclease that cleaves phosphodiester bonds to give mononucleotides and single- or double-stranded oligonucleotides with 5'-phosphate and 3'-hydroxyl ends. Its activity depends on Ca2+ and is further activated by Mg2+ or Mn2+. With Mg2+ it cuts either strand of dsDNA at random sites; with Mn2+ it cuts both strands at nearly the same site, giving blunt ends or 1–2 nt overhangs. The recombinant enzyme is purified from non-animal hosts. Applications include RNA extraction, in vitro transcription, DNA removal before RT-PCR, DNase I footprinting, nick translation and DNA library preparation. For research use only.
Key Features
| RNase-free | Removes DNA without degrading RNA samples. | |
| Concentrated format | Supplied at 50 U/μl with a matching dilution buffer. | |
| Digests ssDNA and dsDNA | Cleaves single- and double-stranded DNA to short oligonucleotides. | |
| Easy inactivation | Stop 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, purified from non-animal hosts |
| Concentration | 50 U/μl |
| Unit definition | One unit completely degrades 1 μg pUC19 plasmid DNA at 37°C in 10 min |
| Cofactors | Ca2+-dependent; activated by Mg2+ or Mn2+ |
| Inactivation | 5 mM EDTA + 65°C for 10 min, or phenol/chloroform extraction |
| Applications | RNA extraction, in vitro transcription, DNA removal before 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 (50 U/μl) | 20 μl | 200 μl |
| DNase I Dilution Buffer | 1 ml | 10 ml |
| 10X Reaction Buffer | 1 ml | 10 ml |
Storage
Store at -30 ~ -15°C.
Before use, dilute the 50 U/μl enzyme to the working concentration (e.g. 1 U/μl) with DNase I Dilution Buffer.
- Remove DNA from RNA before RT-PCR
- In an RNase-free tube, combine:
| Components | Volume |
| RNase-free ddH2O | To 10 μl |
| 10X Reaction Buffer | 1 μl |
| DNase I, RNase-free (diluted to 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 50 mM EDTA to the 10 μl mixture, mix gently and incubate at 65°C for 10 min.
- Use the treated RNA directly as template for RT-PCR.
- Remove template DNA after in vitro transcription
- Add 1 U of diluted DNase I per 0.5 μg template DNA to the transcription reaction.
- Mix gently by pipetting and incubate at 37°C for 15 min.
- Inactivate the DNase I by phenol/chloroform extraction.
Q1: How do I inactivate DNase I after treatment?
- 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: Do I need to dilute the enzyme?
- Yes. The enzyme is supplied at 50 U/μl. Dilute it with the DNase I Dilution Buffer provided to an appropriate working concentration before use.
Q3: What can inhibit DNase I activity?
- Chelating agents, Zn2+, 0.1% SDS, reducing agents such as DTT and mercaptoethanol, and salt concentrations above 50–100 mM.
Q4: How much enzyme should I use 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.
