碧云天生物技术-DNase I(脱氧核糖核酸酶I)(D7076)

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DNase I

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产品编号: D7076

产品包装:1000U

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产品编号	产品名称	产品包装	产品价格
D7073	DNase I	200U	158.00元
D7076	DNase I	1000U	692.00元

DNase I，即Deoxyribonuclease I，中文名称为脱氧核糖核酸酶I，是一种可以消化单链或双链DNA产生单脱氧核苷酸或单链或双链的寡脱氧核苷酸的核酸内切酶。DNase I水解单链或双链DNA后的产物，5'端为磷酸基团，3'端为羟基。
DNase I活性依赖于钙离子，并能被镁离子或二价锰离子激活。镁离子存在条件下，DNase I可随机剪切双链DNA的任意位点；二价锰离子存在条件下，DNase I可在同一位点剪切DNA双链，形成平末端，或1-2个核苷酸突出的粘末端。
特点：不含RNase(RNase free)，可以用于各种RNA样品的处理。提供了用于DNase I失活所需的EDTA。
用途：制备不含DNA的RNA样品；RT-PCR反应前RNA样品中去除基因组DNA等可能的DNA污染；体外T7, T3, SP6等RNA Polymerases催化的RNA转录后去除DNA模板； DNase I footprinting研究DNA-蛋白质相互作用；缺口平移(nick translatioin)；产生DNA随机片段文库；细胞凋亡TUNEL检测中部分剪切基因组DNA作为阳性对照。
来源：从牛胰腺纯化得到。
分子量：约32kDa(单体)。
活性定义：37℃10分钟内，将能够完全降解1μg pBR322质粒DNA所需的酶量定义为1个活性单位。
活性检测条件：40mM Tris-HCl(pH8.0)，10mM MgSO₄，1mM CaCl₂，1μg of pBR322 DNA。
纯度：不含其它DNA内切酶和外切酶，不含RNA酶。
酶储存溶液：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至终浓度为2.5mM后，65℃加热10分钟可使DNase I失活。酚氯仿抽提也可以使DNase I失活。金属离子螯合剂，达到毫摩尔/升浓度的锌离子，0.1%的SDS，DTT、巯基乙醇等还原剂，50-100mM以上盐浓度均对DNase I有显著抑制作用。
包装清单：

产品编号	产品名称	包装
D7076-1	DNase I，RNase-free(50U/μl)	1000U
D7076-2	Reaction Buffer(10X)	1ml
D7076-3	EDTA(25mM)	1ml
－	说明书	1份

保存条件：
-20℃保存。
注意事项：
如需对酶进行稀释，可以用酶储存液(50mM Tris-acetate(pH 7.5)，10mM CaCl₂，50%(v/v)glycerol)进行稀释。
酶使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20℃保存。
本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明：
1.RT-PCR反应前RNA样品中DNA的去除：
a.向一无RNA酶的EP管中依次加入下列试剂：

RNA	1μg
Reaction Buffer (10X)	1μl
补充经DEPC处理的去离子水	至9μl
DNase I(1U/μl)	1μl

注意：如需处理较大量的RNA样品，可以按照比例放大上述反应体系。如果能在上述反应体系中加入适量Ribonuclease inhibitor以防止RNA降解则更佳。
b.37℃孵育30分钟。
c.向上述反应体系中加入1μl 25mM EDTA，65℃孵育10分钟以失活DNase I。注意：在没有鏊合剂如EDTA等存在情况下加热，RNA可被水解。
d.上述加热处理过的RNA样品即可直接用于处理好的RNA可用作RT-PCR反应的模板。
2.体外RNA转录后模板DNA的去除：
a.在每含有0.5μg模板DNA的转录反应体系中加入1U DNase I。注意：在某些情况下，模板DNA完全消化所需的DNase I的量需通过实验进行摸索。
b.37℃ 孵育15分钟。
c.酚/氯仿抽提失活DNase I。
3.缺口平移进行DNA标记：
a.参考下表格设置反应体系：

Reaction Buffer (10X) for DNA Polymerase I	2.5μl
3 dNTP Mixture (1mM each, without the labeled dNTP) *	1.25μl
[α-³²P]-dNTP, ～110TBq/mmol (3000Ci/mmol)	1.85-3.7MBq (50-100μCi)
DNase I (freshly diluted to 0.002U/μl)**	1μl
DNA Polymerase I, E.coli	0. 5-1.5μl (5-15U)
Template DNA	0.25μg
补充无核酸酶去离子水	至25μl

* 3 dNTP Mixture(1mM each, without the labeled dNTP)：分别取除已经标记的dNTP外的3种dNTP(100mM)各1μl加入到97μl 的无核酸酶的去离子水中混匀即可。例如标记的为dATP，则需混合dTTP、dCTP和dGTP三种dNTP至每种的最终浓度为1mM。配制好的dNTP可存放于-20℃以备后续使用。
** DNase I可以用1X Reaction Buffer for DNA Polymerase I进行稀释。
Reaction Buffer(10X)for DNA Polymerase I：500mM Tris-HCl(pH7.5 at 25℃)，100mM MgCl₂，10mM DTT。
b. 立即15℃孵育15～60分钟。
c. 上述反应体系中加入1μl of 0.5M EDTA(pH 8.0)终止反应。
d. 从中取出少量例如1μl检测标记效率。通常标记效率至少可以达到10⁸cpm/μg DNA。
e. 可用Sephadex G-50或Bio-Gel P-60去除[α-³²P]-dNTP，以纯化获得标记的DNA。
4. 其它用途可以参考上述用途进行。

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