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Enzymes

DNase I, RNase-free

Product information

Features

• Recombinant enzyme.
• Purified from non-animal host with a lower level of intrinsic RNases.

Applications

• Preparation of DNA-free RNA (1).
• Removal of template DNA following in vitro transcription (1).
• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR (2).
• DNA labeling by nick-translation in conjunction with DNA Polymerase I (1).
• Studies of DNA-protein interactions by DNase I, RNase-free footprinting (1).
• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn²⁺ is used (3).

Description

DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.

The enzyme activity is strictly dependent on Ca²⁺ and is activated by Mg²⁺ or Mn²⁺ ions, see Fig.1:
– in the presence of Mg²⁺, DNase I cleaves each strand of dsDNA independently, in a statistically random fashion (1);
– in the presence of Mn²⁺, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides (1).

Source

E.coli cells with a cloned gene encoding bovine DNase I.

Molecular Weight

29 kDa monomer.

Definition of Activity Unit

One unit of the enzyme completely degrades 1 µg of plasmid DNA in 10 min at 37°C.

Enzyme activity is assayed in the following mixture: 40 mM Tris-HCl (pH 8.0), 10 mM MgSO₄, 1 mM CaCl₂, 1 µg of pUC19 DNA.
One DNase I unit is approximately equivalent to 0.3 Kunitz unit (4).

Storage Buffer

The enzyme is supplied in:
50 mM Tris-HCl (pH 7.5), 10 mM CaCl₂ and 50% (v/v) glycerol.

10X Reaction Buffer with MgCl₂

100 mM Tris-HCl (pH 7.5 at 25°C), 25 mM MgCl₂, 1 mM CaCl₂.

10X Reaction Buffer without MnCl₂

100 mM Tris-HCl (pH 7.5 at 25°C), 1 mM CaCl₂. Recommended concentration of MnCl₂ in 1X reaction buffer is 10 mM.

Quality Control

The absence of ribonucleases confirmed by appropriate quality test. Functionally tested for digestion of template DNA after in vitro transcription.

Inhibition and Inactivation

• Inhibitors: metal chelators, transition metals (e.g., Zn) in millimolar concentrations, SDS (even at concentrations less than 0.1%), reducing agents (DTT and beta-mercaptoethanol), ionic strength above 50-100 mM.
• Inactivated by heating at 65°C for 10 min in the presence of EGTA or EDTA (use at least 1 mol of EGTA/EDTA per 1 mol of Mn²⁺/Mg²⁺ (5)).

Note

DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.

Figure 1. DNase I activity in the presence of Mg²⁺ or Mn²⁺.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Removal of Template DNA after in vitro Transcription

1. Add 2 u of DNase I, RNase-free per 1 µg of template DNA directly to a transcription reaction mixture. In some cases, the amount of enzyme should be determined empirically.
2. Incubate at 37°C for 15 minutes.
3. Inactivate DNase I by phenol/chloroform extraction.

Removal of Genomic DNA from RNA Preparations

1. Add to an RNase-free tube:
   

   RNA	1 µg
   10X reaction buffer with MgCl2	1 µl
   DNase I, RNase-free	1 µl (1 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

2. Incubate 30 min at 37°C.
3. Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
4. Use the prepared RNA as a template for reverse transcriptase.

Note

• Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
• Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
• RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

Reference

1. Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

Radioactive and Non-radioactive DNA Labeling by Nick-translation

I. Radioactive DNA labeling by nick-translation

1. Mix the following components:

   10X reaction buffer for DNA Polymerase I	2.5 µl
   Mixture of 3 dNTPs, 1 mM* (without the labeled dNTP)	1.25 µl
   [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	1.85-3.7 MBq (50-100 µCi)
   DNase I, RNase-free freshly diluted to 0.002 u/µl**	1 µl
   DNA Polymerase I	0.5-1.5 µl (5-15 u)
   Template DNA	0.25 µg
   Water, nuclease-free	to 25 µl
   Total volume	25 µl

2. Immediately incubate at 15°C for 15-60 minutes.
3. Terminate the reaction by adding 1µl of 0.5 M EDTA, pH 8.0.
4. Take an aliquot (1 µl) to determin the efficiency of the label incorporation. A specific activity of DNA at least 10⁸ cpm/µg DNA is expected.
5. If needed, the labeled DNA may be separated from the unincorporated radioactive precursors on Sephadex G-50 or Bio-Gel P-60 column or using spin column (e.g. GeneJET™ PCR Purification Kit (#K0701).

Note

* To prepare a mixture of three non-labeled dNTPs (1 mM of each), mix 1 µl aliquots of stock solutions of each dNTP (100 mM, from #R0181) with 97 µl of Water, nuclease-free. These dNTP mixes can be stored at -20°C for further use.
** The DNase I, RNase-free can be diluted with the 1X reaction buffer for DNA Polymerase I.

• The reaction volumes can be scaled up or down providing that the final concentrations of the components (DNA, dNTPs, labeled dNTP) are as indicated in the protocol.
• Radioactive DNA probes with higher specific activities can be prepared using two radioactively labeled dNTPs simultaneously. In this case, the composition of the unlabeled dNTP mix should be adjusted accordingly.

II. Non-radioactive DNA labeling by nick-translation
The protocol above can be used for non-radioactive labeling by nick-translation using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

• normal dTTP is subsituted for labeled-dUTP at a molar ratio of 1:3-1:5,
• reaction time is prolonged to 1-2 hours.

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Kienzle, N., et al., DNase I treatment is a prerequisite for the amplification of cDNA from episomal-based genes, BioTechniques, 20, 612-616, 1996.
3. Anderson, S., Shotgun DNA sequencing using cloned DNaseI-generated fragments, Nucleic Acids Res., 9, 3015-3027, 1981.
4. Kunitz, M., J.Gen.Physiol., 33, 349-362, 1950.
5. Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

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