Recombinant DNase I - TAKARA BIO

1 Recombinant DNase I. (RNase-free ). Code No. 2270A. Shipping at 20 . Size 1,000 units Stored at 20 . Supplied 10 DNase I Buffer 1 ml Lot No. Conc. units/ l Volume l Application example : Expiry Date Digestion of genomic DNA in a sample for RNA-PCR. 1. Prepare the following reaction mixture. Total RNA 20 50 g 10 DNase I Buffer (supplied) 5 l Description : Recombinant DNase I (RNase-free) is an endonuclease that Recombinant DNase I (RNase-free) 2 l (10 units). catalyzes to the same extent the degradation of both single- and double- Ribonuclease Inhibitor 20 units stranded DNA randomly,and produces 5'-P terminal oligonucleotides. DEPC-treated water up to 50 l As protease activity has been eliminated, this enzyme is stable around its 2. Incubate for 20 30 min. at 37 . optimum neutral pH range. So, this enzyme is suitable for RNA preparation 3.

2 Perform one of the following procedures to inactivate DNase I. at neutral pH. A Heat treatment (1) Add l of M EDTA, incubate at 80 for 2 min. Storage Buffer : (2) Increase reaction volume to 100 l with DEPC treated 20 mM Tris-HCl, (at 4 ) water. 50 mM NaCl B Phenol/Chloroform extraction mM CaCl2 (1) Mix 50 l of DEPC treated water and 100 l of phenol/. 50 Glycerol chloroform/isoamyl alcohol (25 : 24 : 1) together. (2) Centrifuge at 12,000 rpm for 5 min. at room temperature, Source : Recombinant enzyme derived from non-animal host. then transfer the upper layer to a new tube. (3) Add equal amount of chloroform/isoamyl alcohol (24 : 1). Unit definition : One unit is the amount of the enzyme that increases and mix. the absorbance at 260 nm by per minute at 25 , , with calf (4) Centrifuge at12,000 rpm for 5 min.

3 At room temperature, thymus DNA as the substrate (Kunitz unit).1) then transfer upper layer to new tube. 4. Add 10 l of 3M sodium acetate and 250 l of chilled ethanol, and Reaction mixture for unit definition : then mix. Keep it for 20 min. at 80 . 100 mM Sodium acetate, (at 25 ) 5. Centrifuge at 12,000 rpm for 10 min. at 4 . Remove the supernatant. 5 mM MgSO4 6. Wash the precipitate with chilled 70 ethanol. Centrifuge at 12,000 rpm 40 g/ml calf thymus DNA for 5 min. at 4 and remove the supernatant. 7. Dry the precipitate. Purity : Ribonuclease activity is not detected after incubation of 1 g of 8. Dissolve the precipitate in a suitable amount of DEPC-treated water. 16S, 23S rRNA with 10 units of this enzyme for 4 hours at 37 , Confirm the genomic DNA is removed completely by electrophoresis and measure the RNA concentration.

4 When the genomic DNA is Applications : not removed completely, increase the amount of enzyme or extend 1. For digestion of template DNA without buffer exchange, after in vitro reaction time. transcription with T7 or SP6 RNA ). 2. For nick translation with DNA polymerase I (Cat.#2130)2). References : 3. Making a DNA library for shotgun sequencing in the presence of Mn2+. 3) 1) Kunitz, M. (1950) J. Gen. Physiol ., 33, 349-377. 4. For foot-printing analysis of the interaction between DNA and 2) Rigby, P. W. J.,Dieckmann, M., Rhodes, C. and Berg, P. (1977) J. Mol. ) Biol ., 113. 237-251. 5. For digestion of genomic DNA before RT-PCR. 3) Anderson, S. (1981) Nucleic Acids Res ., 9, 3015-3027. 4) Galas, D. J. and Schmitz, A. (1978) Nucleic Acids Res ., 5, 3157-3170. Note : As protease activity has been eliminated, this enzyme is stable 5) Green, M.

5 R., Maniatis, T. and Melton, D. A. (1983 ) Cell , 32. 681-694. around its optimum neutral pH range. The enzyme requires a divalent metal ion for its activity. It randomly produces nicks in double-stranded Note DNA in the presence of Mg2+, but in the presence of Mn2+, both strands This product is intended to be used for research purpose only. They of double stranded DNA are cleaved into fragments. The enzyme loses its are not to be used for drug or diagnostic purposes, nor are they in- activity reversibly with EDTA, and irreversibly by heat treatment at 80 tended for human use. They shall not to be used products as food, for 10 minutes. cosmetics, or utensils, etc. TAKARA products may not be resold or transfered, modified for resale or Composition of Supplied Reagents ( Store at 20 ) : transfer, or used to manufacture commercial products without writ- 10 DNase I Buffer ten approval from TAKARA BIO INC.

6 400 mM Tris-HCl, If you require licenses for other use, please call at +81 77 543 7247 or 80 mM MgCl2 contact from our website at . 50 mM DTT Produced by TAKARA BIOTECHNOLOGY DALIAN CO., LTD. v200811Da Recombinant DNase I. (RNase-free ). Code No. 2270A. Shipping at 20 . Size 1,000 units Stored at 20 . Supplied 10 DNase I Buffer 1ml 20 . 10 DNase I Buffer 400 mM Tris-HCl, Lot No. ( ). 80 mM MgCl2. Conc. ( ) 50 mM DTT. Volume ( ) . RNA-PCR DNA DNase I . Expiry Date ( ).. 1 . RNA 20 50 g 10 DNase I Buffer 5 l Recombinant DNase I RNase-free DNA Recombinant DNase I RNase-free 2 l 10 U . 5'-P RNase Inhibitor 20 U. endodeoxyribonuclease Mg2+ DNA DEPC 50 l fill up Mn2+ 2 37 20 30 . DNA 3 DNase I . Protease RNase A . RNA 1 l EDTA 80 2 .. 2 DEPC 100 l fill up . 20 mM Tris-HCl, at 4 B / . 50 mM NaCl 1 50 l DEPC 100 l /.

7 MM CaCl2 / 25 24 1 . 50 Glycerol . 2 12,000 rpm 5 . Recombinant enzyme derived from non-animal host.. 3 / 24 1 .. DNA 25 260 nm 4 12,000 rpm 5 . Kunitz unit 1 . 1 1 U . 4 10 l 3M 250 l 80 . 20 . 100 mM , 25 5 4 12,000 rpm 10 . 5 mM MgSO4 6 70 4 12,000rpm 5 . 40 g/ml DNA . 7 . RNase 8 DEPC . 10 U 1 g 16S, 23S rRNA 37 pH 4 DNA . RNA DNase I .. 1 T7 SP6 RNA Polymerase in vitro transcription . RNA buffer DNA 5 1) Kunitz, M. (1950) J. Gen. Physiol ., 33, 349-377. 2) Rigby, P. W. J.,Dieckmann, M., Rhodes, C. and Berg, P. (1977) J. Mol. 2 DNA Polymerase I 2130 nick translation 2 Biol ., 113. 237-251. 3) Anderson, S. (1981) Nucleic Acids Res ., 9, 3015-3027. 3 Mn2+ shot- gun sequencing DNA 3 4) Galas, D. J. and Schmitz, A. (1978) Nucleic Acids Res ., 5, 3157-3170. 5) Green, M. R., Maniatis, T. and Melton, D.

8 A. (1983) Cell , 32. 681-694. 4 DNA- 4 . 5 RT-PCR DNA .. pH Protease .. Mg2+ DNA Mn2+. DNA EDTA . v200811Da 80 10 .. TAKARA Tel 077-543-6116. Fax 077-543-1977.