DNase I，即Deoxyribonuclease I，中文名稱為脫氧核糖核酸酶I，是一種可以消化單鏈或雙鏈DNA產(chǎn)生單脫氧核苷酸或單鏈或雙鏈的寡脫氧核苷酸的核酸內(nèi)切酶。DNase I水解單鏈或雙鏈DNA后的產(chǎn)物，5’端為磷酸基團，3’端為羥基。
DNase I活性依賴于鈣離子，并能被鎂離子或二價錳離子激活。鎂離子存在條件下，DNase I可隨機剪切雙鏈DNA的任意位點；二價錳離子存在條件下，DNase I可在同一位點剪切DNA雙鏈，形成平末端，或1-2個核苷酸突出的粘末端。
Physical Description:? ?Solid
Product Code:? A3778
Product Name:? ?DNase I
Short Description:? ?delivery form: salt-free, freeze-dried powder, chromatographically prepared
Specifications:
Activity (Kunitz): min. 3000 U/mg
Comment:
Deoxyribonuclease I (DNase I) from beef pancreas is an endonuclease (glycoprotein), which preferentially cleaves the phosphodiester bond in the DNA behind pyrimidine nucleotides. This results in a polynucleotide with a 5′-phosphate and a free OH-group in position 3′. DNase I cleaves single-stranded and double-stranded DNA as well as chromatin. The specificity of the enzyme reaction (single-strand-‘Nicks’ versus double-strand breakes) is determined by the ions available. In the presence of Mg2\+?single-strand nicks are generated and in the presence of Mn2\+?double-strand breakes. The pH-optimum of DNase I is 7.8 and it is activated by divalent cations. Maximum activation requires the presence of Mg2\+?and additional Ca2\+. Calcium ions (5 mM) protect DNase I from proteolytic digest. Inhibition is achieved by citrate, if activation is done by magnesium, but not if manganese has been the activator. Besides it is inhibited by chelators such as EDTA and SDS or β-mercaptoethanol.The enzyme is used in molecular biology techniques like digestion of DNA, in the RNA purification (ref. 2 Suppl. 1 pp. 4.1.4) or generating “random nicks” for “nick translation” (ref. 2 Suppl. 9 pp. 3.5.4-6) or ‘footprint’-assays (ref. 2 Suppl. 7 chapter 12.4) or investigations on chromatin (ref. 2 Suppl. 48 chapter 21.4.1).Unit definition\: One unit is defined as that amount of enzyme which causes an increase of absorbance at 260 nm of 0.001 per minute at 25°C based on the method of Kunitz. DNase I is readily soluble in e. g. 0.15 M sodium chloride or in reaction buffer (e. g. 50 mM Tris · Cl, pH 7.5; 10 mM MgCl2?(single-strand ‘nicks’) and 10 mM MnCl2?(double-strand breakes), respecitvely; 50 μg/ml BSA; ref. 2 Suppl. 8 page 3.12.5). For storage dissolve DNase I in 50 % glycerol (w/v); 20 mM Tris · Cl, pH 7.5; 1 mM MgCl2. For stability reasons the concentrations should be at least 1 mg/ml (The maximum solubility is 10 %). This solution is stable for more than one year (ref. 2 Suppl. 8 page 3.12.5). The lyophilized form is stable for 2 – 5 years if kept at \+4°C. If a solution is protease-free, DNase I will not loose significant activity at pH 5 – 7 and 62°C for 5 hours. The enzyme may be heat-inactivated (10 minutes at 99°C).RNase-free DNase I: Dissolve DNase I at 1 mg/ml in 0.1 M iodoacetic acid plus 0.15 M sodium acetate at a final pH of 5.3. The solution is then heated 40 minutes at 55°C and cooled. Finally, 1 M CaCl2?is added to the solution to 5 mM. Store frozen in small aliquots (according to ref. 2 page 3.12.6 Supplement 8).
Bibliography:
(1)Sambrook, J. & Russell, D.W. (2001) Molecular Cloning\: A Laboratory Manual, 3rd Edition. pp. A4.40-42. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2)Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (eds.) 2001. Currrent Protocols in Molecular Biology. John Wiley & Sons, N.Y. (3)McDonald, M.R. (1955) Methods Enzymol. 2, 437-447Deoxyribonucleases (4)Campbell, V.W. & Jackson, D.A. (1980) J. Biol. Chem. 255, 3726-3735The effect of bivalent cations on the mechanism of DNase I. (5)Meinkoth, J. & Wahl, G.M. (1987) Methods Enzymol. 152, 91-94Nick-Translation. (6)Cobianchi, F. & Wilson, S.H. (1987) Methods Enzymol. 152, 94-110Enzymes for modifying and labeling of DNA and RNA.
Storage: -20°C
Origin: from bovine pancreas
產(chǎn)品信息：
| 品牌 |
貨號 |
品名 |
規(guī)格 |
| AppliChem |
A3778,0050 |
DNase I |
50mg |
| AppliChem |
A3778,0100 |
DNase I |
100mg |
| AppliChem |
A3778,0500 |
DNase I |
500mg |
| AppliChem |
A3778_0010 |
DNase I |
10mg |
保存溫度：-20℃，室溫運輸。
應(yīng)用：DNase I 是一種需二價陽離子的脫氧核糖核酸內(nèi)切酶，可用于降解單鏈或雙鏈DNA
用于無DNA污染的RNA的制備，逆轉(zhuǎn)錄及體外轉(zhuǎn)錄等實驗。
產(chǎn)品信息：M=~31000g/mol；CAS-NO:9003-98-9
性狀：凍干粉
配置方法：溶解在150 mM 5毫克/毫升生理鹽水，工作液為無色透明狀。
特點：不含RNase(RNase?free)，可以用于各種RNA樣品的處理。
用途：制備不含DNA的RNA樣品；RT-PCR反應(yīng)前RNA樣品中去除基因組DNA等可能的DNA污 染；體外T7, T3, SP6等RNA Polymerases催化的RNA轉(zhuǎn)錄后去除DNA模板； DNase I footprinting研究DNA-蛋白質(zhì)相互作用；缺口平移(nick?translatioin)；產(chǎn)生DNA隨機片斷文庫；細胞凋亡TUNEL檢 測中部分剪切基因組DNA作為陽性對照。
來源：牛胰腺純化得到。
分子量：約31kDa。
活性檢測條件：40mM Tris-HCl(pH8.0)，10mM MgSO4，1mM CaCl2，1μg of pBR322 DNA。
純度：不含其它DNA內(nèi)切酶和外切酶，不含RNA酶。
酶儲存溶液：50mM Tris-acetate(pH7.5)，10mM CaCl2，50%(v/v)glycerol。
Reaction Buffer(10X)：100mM Tris-HCl(pH7.5 at 25℃)，25mM MgCl2，1mM CaCl2。
失活或抑制：加入EDTA至終濃度為2.5mM后，65℃加熱10分鐘可使DNase I失活。酚氯仿抽提也可以使DNase?I失活。金屬離子螯合劑，達到毫摩爾/升濃度的鋅離子，0.1%的SDS，DTT、巰基乙醇等還原 劑，50-100mM以上鹽濃度均對DNase I有顯著抑制作用。
注意事項：
如需對酶進行稀釋，可以用酶儲存液(50mM?Tris-acetate(pH?7.5)，10mM?CaCl2，50%(v/v)glycerol)進行稀釋。
酶使用時宜存放在冰盒內(nèi)或冰浴上，使用完畢后宜立即放置于-20℃保存。
為了您的安全和健康，請穿實驗服并戴一次性手套操作。
