土壤基因组DNA提取试剂盒      

● 试剂盒内容及保存：

● 储存条件和效期：

本试剂盒在室温（25℃左右）干燥条件下，可保存1年。缓冲液R2和R3可能有沉淀产生，若溶液产生沉淀，应在使用前置于37℃下溶解沉淀至溶液澄清后再使用。

● 产品简介：

土壤样品存在大量的抑制因子如腐殖酸、金属离子等，而纯化的DNA 中只要有这些微量物质的存在，都会影响到PCR等酶促反应。本公司的土壤试剂盒采用独特的腐殖酸去除液（缓冲液R2）能够有效去除腐殖酸；吸附柱CG能能有效去除金属等抑制因子，提纯得到的基因组DNA可直接用于PCR 反应，酶切或定量实验。

● 准备工作：

1. 准备55℃，70℃水浴；无水乙醇；异丙醇；制冰机；1.5ml离心管；2ml离心管

2. 按照标签所示在漂洗缓冲液WB1和漂洗缓冲液PW中加入无水乙醇；缓冲液R5中加入异丙醇，混匀后盖紧瓶盖后室温贮存备用。

3. 每次使用前请检查缓冲液R2，缓冲液R3是否有沉淀生成，如果出现沉淀，37℃温浴至沉淀溶解后再使用。

● 标准操作步骤：

如非指出，所有离心步骤均为使用台式离心机在室温下离心。

1. 称0.3-0.5 g土壤置于2ml离心管中，加入0.4gGlass Beads，再加入700 μl 缓冲液R1与100 μl 缓冲液R2。涡旋器高速震荡3-5min。

注意：对含水量丰富的样品，可以预先离心除去部分水分后再称取样品。缓冲液R2是腐殖酸去除剂，100μl对大部分样品来说足以有效除去腐殖酸等抑制因子。对一些腐殖酸含量特别丰富的土壤， 缓冲液R2的量可以适当增加，但不能超过250μl，否则会严重影响DNA的得率。

2. 加入100 μl 缓冲液R3（R3如有沉淀37℃水浴完全溶解后再用），涡旋混匀。70℃水浴处理10min。期间振荡几次。注意：如果要纯化革兰氏阳性菌的DNA，请在70℃处理完后，再90℃水浴处理2min。

3. 12000 rpm(~13,000g)离心1分钟,取600μl上清到1.5ml离心管中，加入180 μl 缓冲液R4混匀。

注：转移上清时确保不要吸取到沉淀，转移的上清量最好不超过80%。

4. 冰上放置5分钟。12000 rpm(~13,000g)离心1分钟。 转移上清到新的1.5ml离心管中。

注：转移上清时确保不要吸取到沉淀，转移的上清量最好不超过80%。

5. 加入与上清等体积的缓冲液R5（使用前请检查是否已加入异丙醇），颠倒混匀。

6. 取750 μl混合液加到吸附柱中（吸附柱放入收集管中），12000 rpm(~13,000g)离心30秒，倒掉滤出液。

7. 将剩余混合液加到吸附柱中(吸附柱放入收集管中),12000rpm(~13,000g)离心30秒,倒掉滤过液。

8. 向吸附柱CG中加入500 μl 漂洗缓冲液WB1(使用前请先检查是否已加入无水乙醇),12,000 rpm (~13,000×g )  离心30 秒，倒掉废液。

9. 向吸附柱CG中加入600 μl 漂洗缓冲液PW(使用前请先检查是否已加入无水乙醇)，12,000 rpm (~13,000g) 离心30 秒，倒掉废液，吸附柱CG放入收集管中。

10. 向吸附柱CG中加入600 μl漂洗缓冲液PW，12,000 rpm (~13,000g) 离心30秒，倒掉废液，将吸附柱CG放入收集管中。

11. 12,000 rpm(~13,000g)离心2分钟，以彻底晾干吸附材料中残余的漂洗液。

注：此步骤非常重要，其目的是将吸附柱中残余的漂洗液去除。漂洗液中乙醇的残留会影响后续的酶反应（酶切、PCR等）实验。

12. 将吸附柱CG转入一个干净的1.5ml离心管中，向吸附膜的中间部位悬空滴加50–100 μl经70℃水浴预热的洗脱缓冲液EB，室温放置2分钟，12,000 rpm(~13,000g)离心2分钟。

注:  = 1 \* GB3 ① 洗脱缓冲液体积最好不少于50 μl，体积过小影响回收效率。

= 2 \* GB3 ② 洗脱液的pH值对于洗脱效率有很大影响。若用水做洗脱液应保证其pH值在7.0-8.5范围内，pH值低于7.0会降低洗脱效率。

13. DNA产物-20℃保存。

大量操作步骤（针对含微量核酸的样品）

1. 称1-5 g土壤置于10ml离心管中，加入1gGlass Beads，再加入3ml 缓冲液R1与200μl 缓冲液R2。涡旋器高速震荡3-5分钟。

2. 加入600μl 缓冲液R3，涡旋混匀。70℃水浴处理10分钟。期间振荡几次。

3.3000g离心3分钟，转移上清到新的离心管中，加入550μl 缓冲液R4混匀。

4. 冰上放置5分钟。8000 g离心10分钟。转移上清到新的离心管中。

5. 以下按标准操作步骤的第五步继续操作。

● DNA浓度及纯度检测：

基因组DNA片段的大小与样品保存时间、操作过程中的剪切力等因素有关。提取的DNA片段可用琼脂糖凝胶电泳和紫外分光光度计检测浓度与纯度。可配制0.8-1.0％琼脂糖凝胶，使用λ/HindIII判断基因组的大小，完整的基因组大小应在23kb以上。使用分光光度计检测时， OD₂₆₀/OD₂₈₀比值应为1.7–1.9之间，如果洗脱时不使用洗脱缓冲液，而使用去离子水洗脱，比值可能偏低，但并不表明DNA纯度不高。

● 常见问题分析：

 实验示例：

Buffer R2去除腐殖酸效果对比图
左图-步骤1提取时使用了Brffer R2，颜色明显浅很多(左1,左2)，
说明Buffer R2对去除腐殖酸、色素等效果明显
右图-步骤3上清加入Buffer R4进一步处理，离心去除杂质，
裂解时加有Buffer R2的，再经Buffer R4处理几乎不再有颜色(右1,右2)，
而不加Buffer R2的颜色还很深(右3，右4)。

1, 2: SDS高盐酚氯仿抽提法
3, 4: 土壤基因组DNA提取试剂盒(RTG2403)提取
1, 3: 为花基土壤
2, 4: 河边淤泥

核酸提取产品发表文章

1. [2008 IF=1.749] Development of a sequence-characterized ampli?ed region marker for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kuhn.

Author: J.H. Liu, L. Gao, T.G. Liu and W.Q. Chen

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: Letters in Applied Microbiology 2009,49,235-240

Institution：Institute of Plant Protection ,Chinese Academy of Agricultural Sciences

Paper link： https://doi.org/10.1111/j.1472-765X.2009.02645.x

2. [2009 IF=2.435] Characterization of three new S-alleles and development of an S-allele-specific PCR system for rapidly identifying the S-genotype in apple cultivars.

Author: Shenshan Long, Maofu Li, Zhenhai Han, Kun Wang, Tianzhong Li

Product: RTP2201琼脂糖凝胶回收试剂盒

Journal: Tree Genetics & Genomes (2010) 6:161–168

Institution：China Agricultural University

Paper link： https://link.springer.com/article/10.1007/s11295-009-0237-6

3. [2010 IF=0.921] An ISSR-based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat, Caused by Tilletia controversa Kuhn.

Author: Li Gao,Wanquan Chen and Taiguo Liu

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: J Phytopathol 159:155–158 (2011)

Institution：State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, CAAS

Paper link：https://onlinelibrary.wiley.com/doi/10.1111/j.1439-0434.2010.01735.x

4. [2010 IF=1.359] Curing the Plasmid pXO2 from Bacillus anthracis A16 Using Plasmid Incompatibility.

Author: Huagui Wang, Xiankai Liu, Erling Feng, Li Zhu, Dongshu Wang, Xiangru Liao, Hengliang Wang

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: Curr Microbiol (2011) 62:703–709

Institution：State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology

Paper link：https://link.springer.com/article/10.1007/s00284-010-9767-2

5. [2010 IF=1.993] Computation-assisted SiteFinding-PCR for isolating flanking sequence tags in rice

Author: Hongru Wang, Jun Fang, Chengzheng Liang, Minghui He, Qiye Li, and Chengcai Chu

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: BioTechniques Vol. 51 | No. 6 | 2011

Institution：Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Paper link：https://pubmed.ncbi.nlm.nih.gov/22150334/

6. [2012 IF=0.903] T Polymorphisms in major histocompatibility complex class IIa genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792).

Author: Z. Liu, D.-D. Hu, S.-J. Shao, J.-Q. Huang, J.-F. Wang and J. Yang

Product: RTP2202 PCR产物纯化试剂盒

Journal: J. Appl. Ichthyol. 29 (2013), 1234–1240

Institution：College of Animal Science and Technology, Gansu Agricultural University

Paper link：https://onlinelibrary.wiley.com/doi/full/10.1111/jai.12326

7. [2012 IF=1.958] Cloning, bioinformatics and the enzyme activity analyses of a phenylalanine ammonia-lyase gene involved in dragon’s blood biosynthesis in Dracaena cambodiana.

Author: Xing-Hong Wang，Changhe Zhang

Product: RTP2202 PCR产物纯化试剂盒

Journal: Mol Biol Rep (2013) 40:97–107

Institution：Yunnan Institute of Microbiology, Yunnan University

Paper link：https://link.springer.com/article/10.1007/s11033-012-2032-y

8. [2013 IF=1.687] Tobacco Arabinogalactan Protein NtEPc Can Promote Banana (Musa AAA) Somatic Embryogenesis.

Author: H. Shu & L. Xu & Z. Li & J. Li & Z. Jin & S. Chang

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: Appl Biochem Biotechnol (2014) 174:2818–2826

Institution：Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences

Paper link：https://link.springer.com/article/10.1007/s12010-014-1228-0

9. [2015 IF=1.32] Association between MHC II beta chain gene polymorphisms and resistance to infectious haematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum, 1792).

Author: Juan Yang, Zhe Liu, Hai-Na Shi, Jiu-Pan Zhang, Jian-Fu Wang, Jin-Qiang Huang & Yu-Jun Kang

Product: RTP2202 PCR产物纯化试剂盒

Journal: Aquaculture Research, 2016, 47, 570–578

Institution：College of Animal Science and Technology, Gansu Agricultural University

Paper link： https://onlinelibrary.wiley.com/doi/10.1111/are.12516

10. [2015 IF=] A weird DNA band in PCR and its cause.

Author: Chang Shenghe, Sun Wei, Zhou Zhaoxi, Li Jingyang, Dai Minjie, Shu Haiyan

Product: RTP2102 普通质粒小提试剂盒

Journal: Journal of Plant Science & Molecular Breeding  Volume 5 Article 2 2016

Institution：Haikou experimental station, Chinese Academy of Tropical Agricultural Sciences

Paper link：

11. [2017 IF=4.076] Application of Real-Time Quantitative PCR to Detect Mink Circovirus in Naturally and Experimentally Infected Minks.

Author: Xingyang Cui, Yunjia Shi, Lili Zhao, Shanshan Gu, Chengwei Wei, Yan Yang,Shanshan Wen, Hongyan Chen and Junwei Ge

Product: RTP2102 普通质粒小提试剂盒

Journal: Fronties in Microbiology May 2018 | Volume 9 | Article 937

Institution：College of Veterinary Medicine, Northeast Agricultural University

Paper link：https://pubmed.ncbi.nlm.nih.gov/29867846

12. [2017 IF=3.974] In vitro and in vivo toxic e?ects and in?ammatory responses induced by carboxylated black carbon-lead complex exposure.

Author: Shuanglin Jiang,, Mengting Shang,Kui Mu，Nan Jiang，Haiyan Wen，Rong Wang，Hai Wu，Wenyong Li

Product: RTG2402 动物/细胞基因组DNA提取试剂盒

Journal: Ecotoxicology and Environmental Safety 165 (2018) 484–494

Institution：Key Laboratory of Embryo Development and Reproductive Regulation of Anhui Province, Fuyang Normal University

Paper link：http://www.sciencedirect.com/science/article/pii/S0147651318309011

13. [2018 IF=8.063] ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma

Author: Jie Luo,# Yuanzheng Xia,# Yong Yin, Jun Luo, Mingming Liu, Hao Zhang, Chao Zhang, Yucheng Zhao, Lei Yang, and Lingyi Kong

Product: RTP2101 高纯质粒小提试剂盒

Journal: Theranostics 2019, Vol. 9, Issue 21

Institution：School of Traditional Chinese Pharmacy, China Pharmaceutical University

Paper link：https://pubmed.ncbi.nlm.nih.gov/31534554

14. [2020 IF=1.857] Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae).

Author: Yakai Tian, Yingping Qu,Kun Dong,Shaoyu He,Wu Jie, and Jiaxing Huang

Product: RTP2201 琼脂糖凝胶回收试剂盒

Journal: Journal of Insect Science (2021) 21(5): 13; 1–8

Institution：Institute of Apicultural Research, Chinese Academy of Agricultural Sciences

Paper link：https://doi.org/10.1093/jisesa/ieab078

15. [2020 IF=1.336] Isopentenyl Diphosphate Isomerase (IPI) Gene Silencing Negatively Afects Patchouli Alcohol Biosynthesis in Pogostemon cablin

Author: Wuping Yan, Yuzhang Yang, Yougen Wu, Jing Yu, Junfeng Zhang, Dongmei Yang, Zeeshan Ul Haq Muhammad

Product: RTP2102 普通质粒小提试剂盒

Journal: Plant Molecular Biology Reporter Published 27 January 2021

Institution：College of Horticulture, Hainan University

Paper link：https://link.springer.com/article/10.1007/s11105-020-01269-0

16. [2021 IF=6.064] Genome resequencing and transcriptome analysis reveal the molecular mechanism of albinism in Cordyceps militaris.

Author: Ying Zhao, YuDong Liu, Xun Chen and Jun Xiao

Product: RTG2407 真菌基因组DNA提取试剂盒

Journal: Fronties in Microbiology.  Published 11 April 2023

Institution： Institute of Edible Fungi, Liaoning Academy of Agricultural Sciences

Paper link：https://www.frontiersin.org/articles/10.3389/fmicb.2023.1153153/full