DNaseI, RNase-free (500 u)

Concentration: 1u/μl                                                                                                                  Cat. No.: PR891627                                                                                                                                          

20-Supplied with: 1 ml of 10X Reaction Buffer with MgCl2, 1 ml of 50 mM EDTA                         Store at 

Description: 

DNaseI is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono and oligodeoxyribo- nucleotides with 5’-phosphate and 3’-OH groups. The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions: 

⦁    In the presence of Mg2+, DNaseI cleaves each strand of dsDNA independently, in a statistically random fashion;

⦁    In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt ends or with one or two nucleotide overhangs .

Applications:

⦁    Preparation of DNA-free RNA.

⦁    Removal of template DNA following in vitro transcription, see protocol on reverse page.

⦁    Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR , see protocol on reverse page.

⦁    DNA labeling by nick-translation in conjunction with DNA Polymerase I, see protocol on reverse page.

⦁    Studies of DNA-protein interactions by DNaseI, RNase-free footprinting .

⦁    Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is used .

Source:

E.coli cells with a cloned gene encoding bovine DNaseI. 

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: 10 mM Tris-HCl (pH 7.5 at 25°C), 2.5 mM MgCl2, 0.1 mM CaCl2, 1 μg of pUC19 DNA. 

One DNaseI unit is equivalent to 0.3 Kunitz unit. 

Storage Buffer 

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. 

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 2- 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+). 

Note

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

Removal of genomic DNA from RNA preparations 

1.    Add to an RNase-free tube: 

RNA     1 μg 

10X reaction buffer with MgCl2     1 μl 

DNaseI, RNase-free     0.5μl (0.5u) 

DEPC-treated Water     to 10 μl 

2. Incubate at 37°C for 30 min. 

3. Add 1 μl 50 mM EDTA and incubate at 65°C for 10 min. RNA hydrolyzes during heating with divalent cations in the absence of a chelating agent. Alternatively, use phenol/chloroform extraction. 

4. Use the prepared RNA as a template for reverse transcriptase. 

Note 

⦁    Do not use more than 1 u of DNaseI, RNase-free per 1 μg of RNA. 

⦁    Volumes of the reaction mixture and 50 mM EDTA solution can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1 μg/μl. 

⦁    RiboLock™ RNase Inhibitor, typically at 1 u/μl, can also be included in the reaction mixture to prevent RNA degradation. 

Removal of template DNA after in vitro transcription 

1. Add 2u of DNaseI, 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 DNaseI by phenol/chloroform extraction.

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 each (without the labeled dNTP)    1.25 μl

[32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)    1.85-3.7 MBq (50-100 μCi)

DNaseI, RNase-free freshly diluted to 0.002 u/μl    0.5 μl

DNA Polymerase I Template DNA    0.5-1.5 μl (5-15u) 0.25 μg

Water, nuclease-free    to 25 μl

2. Immediately incubate at 15°C for 15-60 min. 

3. Terminate the reaction by adding 1 μl of 0.5 M EDTA, pH 8.0. 

 4. Take an aliquot (1 μl) to determine the efficiency of label incorporation. A specific activity of at least 10 8cpm/μg DNA is expected.

Note

⦁    DNaseI, RNase-free can be diluted with 1X reaction buffer for DNA Polymerase I: 50 mM Tris-HCl (pH 7.5 at 25°C), 10 mM MgCl2 and 1 mM DTT

Quality Control Assay Data

No degradation of RNA was observed after incubation of 5 units of DNaseI with 160 ng RNA for 4 hours at 37°C.