Description
DNase I, or Deoxyribonuclease I, is an endonuclease that degrades single- or double-stranded DNA to produce single deoxynucleotides or single- or double-stranded oligodeoxynucleotides. The produced short oligos have 5′-phosphorylate and 3′-OH.
DNase I activity is calcium-dependent and can be activated by magnesium ions or divalent manganese ions. In the presence of magnesium ions, DNase I can randomly cleave double-stranded DNA at any sites. In the presence of divalent manganese ions, DNase I cleaves the double-strands at the same site to generate blunt ends or sticky ends with 1-2 nucleotide overhangs.
Features: This product is RNase free and can be used for processing various RNA samples. EDTA required for DNase I inactivation is also provided in this product.
Applications: Preparation of RNA without DNA; removal of DNA from RNA samples for RT-PCR reactions; removal of DNA templates after in vitro RNA transcription catalyzed by RNA Polymerases such as T7, T3, SP6; DNase I footprinting to study DNA-Protein interactions; nick translation; generation of DNA random fragment libraries; partial cleavage of genomic DNA in apoptosis TUNEL assay as a positive control.
Source: Purified from bovine pancreas.
Molecular weight: ~32kDa (monomer).
Definition of activity: One unit is defined as the amount of enzyme required to completely degrade 1µg of pBR322 plasmid DNA within 10 minutes at 37 ℃.
Activity assay condition: 40mM Tris-HCl (pH 8.0), 10mM MgSO4 , 1mM CaCl2 , 1µg of pBR322 DNA.
Purity: Free of RNase and other DNA endonucleases and exonucleases.
Enzyme storage buffer: 50mM Tris-acetate (pH 7.5), 10 mM CaCl2, 50% (v/v) glycerol.
Reaction Buffer (10X): 100mM Tris-HCl (pH7.5 at 25 ℃ ), 25mM MgCl2 , 1mM CaCl2 .
Inactivation or inhibition: DNase I is inactivated by incubation at 65℃ for 10 minutes after adding EDTA to a final concentration of 2.5mM. Phenol-chloroform extraction can also inactivate DNase I. Metal ion chelators, zinc ions at a concentration of mmol/L, 0.1% SDS, DTT, mercaptoethanol and other reducing agents, and salt concentrations above 50-100mM all have a significant inhibitory effect on DNase I.
Packing List：
| Item |
Component |
Quantity |
| D1037-1 |
DNase I, RNase-free (1U/µl) |
200U |
| D1037-2 |
Reaction Buffer (10X) |
0.2ml |
| D1037-3 |
EDTA (25mM) |
0.2ml |
| Manual |
– |
1 copy |
Storage Conditions：
Store at -20℃.
Precautions：
DNase I should be kept on ice when handling, and should be stored at -20 ℃ immediately after use.
This product is for R&D only. Not for drug, household, or other uses.
For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instruction
Instructions for Use：
1. Removal of DNA from RNA samples for RT-PCR
a. To an RNase-free microcentrifuge tube, add the following reagents in sequence:
| Component |
Volume |
| RNA |
1µg |
| Reaction Buffer (10X) |
1µl |
| Nuclease-free Water |
to 9µl |
| DNase I (1U/µl) |
1µl |
Note: The reaction mixture can be scaled up to process larger amounts of RNA samples. It is recommended to add an appropriate amount of Ribonuclease inhibitor to prevent RNA degradation.
a. Incubate at 37℃ for 30 minutes.
b. Add 1µl of 25mM EDTA, mix well, and incubate at 65℃ for 10 minutes to inactivate DNase I.
Note: RNA can be hydrolyzed at high temperatures in the absence of chelators.
c. The product can be used directly in RT-PCR reactions.
2. Removal of template DNA after RNA transcription in vitro
a. Add 1 U of DNase I to each transcription reaction containing 0.5µg of template DNA.
Note: In some cases, the amount of DNase I required for complete digestion of template DNA needs to be determined by testing.
b. Incubate at 37 ℃ for 15 minutes.
c. Remove DNase I by phenol/chloroform extraction.
3. Nick translation for DNA labeling
a. Set up the following reaction on ice:
| Component |
Volume |
| 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 |
| Nuclease-free Water |
to 25µl |
*3 dNTP Mixture (1mM each, without the labeled dNTP): Take 1µl of each of the three dNTPs (100mM) except the labeled dNTP and add them to 97µl of Nuclease-free Water and mix well. For example, if dATP is labeled, dTTP, dCTP and dGTP, need to be mixed to a final concentration of 1mM each. Prepared dNTPs can be stored at -20℃ for future use.
**DNase I can be diluted with 1X Reaction Buffer for DNA Polymerase I.
Reaction Buffer (10X) for DNA Polymerase I: 500mM Tris-HCl (pH7.5 at 25℃), 100mM MgCl2, 10mM DTT.
b. Immediately incubate at 15℃ for 15-60 minutes.
c. Add 1µl of 0.5M EDTA (pH 8.0) to stop the reaction.
d. Take a small amount (e.g., 1µl) of reaction product to check the labeling efficiency. Usually, the labeling efficiency can reach at least 10 8 cpm/µg DNA.
e. [α-32P]-dNTP can be removed with Sephadex G-50 or Bio-Gel P-60 to purify labeled DNA.
4. For other uses, please refer to the protocol above or other related literature.
References
Related papers：
1. Xiang Shi, Yanrong Zhuang, Zhenhua Chen, Mingrui Xu, Junqi Kuang, Xue-Lian Sun, Lisen Gao, Xia Kuang, Huairen Zhang, Wei Li, Samuel Zheng Hao Wong, Chuanyu Liu, Longqi Liu, Danhua Jiang, Duanqing Pei, Yi Lin, Qing-Feng Wu
.
Sci Adv. 2022 Nov 18;8(46):eabq2987. doi: 10.1126/sciadv.abq2987. (IF 13.116)
2. Qiao C, Liu J, Yang J, Li Y, Weng J, Shao Y, Zhang X.
.
Biomaterials. 2016 Apr;85:1-17. (IF 10.317)
3. Li H, Zhang B, Lu X, Tan X, Jia F, Xiao Y, Cheng Z, Li Y, Silva DO, Schrekker HS, Zhang K, Mirkin CA.
.
P NATL ACAD SCI USA. 2018 Apr 24 115(17):4340-4344. (IF 9.412)
4. Wen Li, Zhuo Luo, Chang-Yu Yan, Xiao-Hua Wang, Zheng-Jie He, Shu-Hua Ouyang, Chang Yan, Li-Fang Liu, Qing-Qing Zhou, Han-Lu Mu, Hai-Biao Gong, Wen-Jun Duan, Lei Liang, Hiroshi Kurihara, Du Feng, Yi-Fang Li, Rong-Rong He
.
Theranostics. 2020 Jul 11;10(20):9032-9049. doi: 10.7150/thno.46921. (IF 8.579)
5. Xiao-Long Mo, Feng Liu, Chun-Hua Xing, Meng-Meng Shan, Bo Yao, Qi-Qi Sun, Yuan-Jing Zou, Kun-Huan Zhang, Jun Tan, Shao-Chen Sun, Yan-Ping Ren
.
J Cell Physiol. 2022 Dec;237(12):4580-4590. doi: 10.1002/jcp.30910. (IF 5.546)
6. Peng C, Rao W, Zhang L, Gao F, Hui H, Wang K, Dai S, Yang Y, Luo P, Ma Y, Ma W, Yu X, Fei Z
.
CELL PHYSIOL BIOCHEM. 2018 46(6):2311-2324. (IF 5.5)
7. Li J, Mao Q, He J, She H, Zhang Z, Yin C.
.
Stem Cell Res Ther. 2017 Mar 9;8(1):55. (IF 5.116)
8. Wang H, Wu J.
.
Mol Med Rep. 2016 Mar;13(3):2892-8. (IF 2.1)
9. Hui H, Rao W, Zhang L, Xie Z, Peng C, Su N, Wang K, Wang L, Luo P, Hao YL, Zhang S, Fei Z.
.
Neurochem Int. 2016 Mar;94:23-31. (IF 3.881)
10. Zhu W, Xie K, Xu Y, Wang L, Chen K, Zhang L, Fang J.
.
Virus Res. 2016 Jun 2;217:125-32. (IF 2.934)
11. Yang S, Li SS, Yang XM, Yin DH, Wang L.
.
Oncol Lett. 2016 Jun;11(6):4167-4176. (IF 2.311)
12. Zhu G, Chen J, Tian J, Ge L, Xing A, Tang G.
.
Mol Med Rep. 2016 Jul;14(1):509-14. (IF 2.1)
13. Feng Q, Hu ZY, Liu XQ, Zhang X, Lan X, Geng YQ, Chen XM, He JL, Wang YX, Ding YB.
.
Reprod Biomed Online. 2016 Nov 23. pii: S1472-6483(16)30614-9. (IF 3.218)
14. Sun XY, Duan ZJ, Liu Z, Tang SX, Li Y, He SC, Wang QM, Chang QY.
.
Exp Ther Med. 2016 Dec;12(6):3716-3722. (IF 1.785)
15. Zhang Q, Cheng G, Qiu H, Wang Y, Wang J, Xu H, Zhang T, Liu L, Tao Y, Ren Z.
.
Exp Ther Med. 2017 Aug;14(2):1795-1801. (IF 1.785)
16. Liu X, Tian F, Wang S, Wang F, Xiong L.
.
REJUV RES. 2017 Dec 22. doi: 10.1089/rej.2017.1999. [Epub ahead of print] (IF 3.811)
17. Pan Z, Fan Z, Ma J, Liu H, Shen L, He B, Zhang M
.
Am J Transl Res. 2019 Jun 15 11(6):3801-3815. eCollection 2019. (IF 3.375)
18. Zhao RZ, Jiang S, Ru NY, Jiao B, Yu ZB
.
CAN J PHYSIOL PHARM. 2019 Oct 97(10):980-988. (IF 1.946)
19. Zhong J, Xu W
.
Exp Ther Med. 2019 Nov 18(5):4002-4010. (IF 1.785)
20. Jiang Zhong, Wei Xu
.
Exp Ther Med. 2019 Nov;18(5):4002-4010. doi: 10.3892/etm.2019.8056. (IF 1.785)
21. Jin Liu, Yong-Ming Zhu, Yi Guo, Liang Lin, Zhan-Xiang Wang, Feng Gu, Xin-Yi Dong, Ming Zhou, Yi-Fan Wang, Hui-Ling Zhang
.
Front Pharmacol. 2020 Jun 12;11:812. doi: 10.3389/fphar.2020.00812. (IF 4.225)
22. Jian Tan, Zhiguo Wu, Jun Liu, Wenting Zhang, Wanqiu Yuan, Hong Peng
.
J Cell Mol Med. 2020 Sep;24(17):9825-9838. doi: 10.1111/jcmm.15566. (IF 4.486)
23. Qiang Yang, Yanghao Zhou, Jin Chen, Ning Huang, Zhigang Wang, Yuan Cheng
.
Int J Nanomedicine. 2021 Jan 8:16:185-199. doi: 10.2147/IJN.S286221.
24. Fenju Qin, Ningang Liu, Jing Nie, Tao Shen, Yingjie Xu, Shuxian Pan, Hailong Pei, Guangming Zhou
.
Environ Health Prev Med. 2021 Oct 11;26(1):103. doi: 10.1186/s12199-021-01021-4.
25. Jiwen Xu, Xiaoyan Liu, Xueting Liu, Yunlai Zhi
.
Bioengineered. 2021 Dec;12(1):6687-6702. doi: 10.1080/21655979.2021.1973775. (IF 2.205)
26. Xi Chen, Yunxia Liu, Dongyuan Sun, Rongqi Sun, Xiaoxiao Wang, Minmin Li, Ning Song, Jicheng Ying, Tao Guo, Yingying Jiang
.
Cancer Sci. 2022 Jun;113(6):2071-2084. doi: 10.1111/cas.15366. (IF 4.966)
27. Bao-Fei Yan, Xi Chen, Ya-Fang Chen, Sheng-Jin Liu, Chen-Xin Xu, Ling Chen, Wen-Bo Wang, Ting-Ting Wen, Xian Zheng, Jia Liu
.
J Ethnopharmacol. 2022 Aug 10:294:115365. doi: 10.1016/j.jep.2022.115365. (IF 3.69)
28. Yongxiao Yang, Zhengmei Li, Qin Li, Kai Ma, Yan Lin, Huigen Feng, Tianyun Wang
.
Appl Microbiol Biotechnol. 2022 Aug;106(13-16):4963-4975. doi: 10.1007/s00253-022-12051-5.
29. Jing Zheng, Wenyuan Chen, Hao Xiao, Fan Yang, Xiaowu Li, Jingdong Song, Lingpeng Cheng, Hongrong Liu
.
Viruses. 2022 Nov 1;14(11):2431. doi: 10.3390/v14112431.
30. Longhai Du, Guanghao Zhu, Yanlong Xu, Binxu Han, Yu Wang, Minhui Zhu, Yingdi Meng, Huaiwen Chen, Zuochong Yu
.
Front Bioeng Biotechnol. 2023 Feb 17:11:1147064. doi: 10.3389/fbioe.2023.1147064.
31. Jianpu Tang, Xuemei Jia, Qian Li, Zhen Cui, Aiqi Liang, Bin Ke, Dayong Yang, Chi Yao
.
Proc Natl Acad Sci U S A. 2023 Jul 11;120(28):e2303822120. doi: 10.1073/pnas.2303822120.
32. Yao Zhang, Xiao-Qiu Zhou, Wei-Dan Jiang, Pei Wu, Yang Liu, Hong-Mei Ren, Xiao-Wan Jin, Lin Feng
.
Int J Mol Sci. 2023 Jul 8;24(14):11243. doi: 10.3390/ijms241411243.
33. Jingyi Cui, Lanxin Zhang, Zaifeng Zhang, Xuanmei Luo, Ye Liu, Chang Li, Wei Huang, Lihui Zou, Xue Yu, Fei Xiao
.
Nucleic Acids Res. 2023 Aug 11;51(14):e78. doi: 10.1093/nar/gkad554.
34. Youqiang Fang, Xing Luo, Yanteng Xu, Zheng Liu, Rachel L Mintz, Haiyang Yu, Xuan Yu, Kai Li, Enguo Ju, Haixia Wang, Zhaohui Tang, Yu Tao, Mingqiang Li
.
Adv Sci (Weinh). 2023 Aug;10(22):e2300899. doi: 10.1002/advs.202300899.
35. Yurong Zhu, Jun Li, Tao Ba, Yuan Sun, Xiangyun Chang
.
Autoimmunity. 2023 Dec;56(1):2194584. doi: 10.1080/08916934.2023.2194584. (IF 2.125)
36. Haiyan Mai, Junjian Li, Yuyan Luo, Jiayi Ou, Gong Chen, Lianbao Ye
.
Chem Biodivers. 2023 Dec;20(12):e202301111. doi: 10.1002/cbdv.202301111.
37. Haotian Chen, Donghui Shi, Changfeng Guo, Wentao Zhang, Yadong Guo, Fuhan Yang, Ruiliang Wang, Junfeng Zhang, Zujun Fang, Yang Yan, Shiyu Mao, Xudong Yao
.
Mol Carcinog. 2024 Mar;63(3):461-478. doi: 10.1002/mc.23664.
38. Yinfeng Xu, Chuying Qian, Qian Wang, Lijiang Song, Zhengfu He, Wei Liu, Wei Wan
.
Autophagy. 2024 May;20(5):1134-1146. doi: 10.1080/15548627.2023.2287932.
39. Deming Ou, Ying Wu, Jibin Zhang, Jun Liu, Zeyu Liu, Minfeng Shao, Xiaoying Guo, Shiman Cui
.
Biochem Genet. 2024 Jun;62(3):1657-1674. doi: 10.1007/s10528-023-10484-9.
