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土壤基因組DNA提取試劑盒      

● 試劑盒內容及保存：

● 儲存條件和效期：

本試劑盒在室溫（25℃左右）干燥條件下，可保存1年。緩沖液R2和R3可能有沉淀產生，若溶液產生沉淀，應在使用前置于37℃下溶解沉淀至溶液澄清后再使用。

● 產品簡介：

土壤樣品存在大量的抑制因子如腐殖酸、金屬離子等，而純化的DNA 中只要有這些微量物質的存在，都會影響到PCR等酶促反應。本公司的土壤試劑盒采用獨特的腐殖酸去除液（緩沖液R2）能夠有效去除腐殖酸；吸附柱CG能能有效去除金屬等抑制因子，提純得到的基因組DNA可直接用于PCR 反應，酶切或定量實驗。

● 準備工作：

1. 準備55℃，70℃水??；無水乙醇；異丙醇；制冰機；1.5ml離心管；2ml離心管

2. 按照標簽所示在漂洗緩沖液WB1和漂洗緩沖液PW中加入無水乙醇；緩沖液R5中加入異丙醇，混勻后蓋緊瓶蓋后室溫貯存?zhèn)溆谩?

3. 每次使用前請檢查緩沖液R2，緩沖液R3是否有沉淀生成，如果出現(xiàn)沉淀，37℃溫浴至沉淀溶解后再使用。

● 標準操作步驟：

如非指出，所有離心步驟均為使用臺式離心機在室溫下離心。

1. 稱0.3-0.5 g土壤置于2ml離心管中，加入0.4gGlass Beads，再加入700 μl 緩沖液R1與100 μl 緩沖液R2。渦旋器高速震蕩3-5min。

注意：對含水量豐富的樣品，可以預先離心除去部分水分后再稱取樣品。緩沖液R2是腐殖酸去除劑，100μl對大部分樣品來說足以有效除去腐殖酸等抑制因子。對一些腐殖酸含量特別豐富的土壤， 緩沖液R2的量可以適當增加，但不能超過250μl，否則會嚴重影響DNA的得率。

2. 加入100 μl 緩沖液R3（R3如有沉淀37℃水浴完全溶解后再用），渦旋混勻。70℃水浴處理10min。期間振蕩幾次。注意：如果要純化革蘭氏陽性菌的DNA，請在70℃處理完后，再90℃水浴處理2min。

3. 12000 rpm(~13,000g)離心1分鐘,取600μl上清到1.5ml離心管中，加入180 μl 緩沖液R4混勻。

注：轉移上清時確保不要吸取到沉淀，轉移的上清量最好不超過80%。

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

注：轉移上清時確保不要吸取到沉淀，轉移的上清量最好不超過80%。

5. 加入與上清等體積的緩沖液R5（使用前請檢查是否已加入異丙醇），顛倒混勻。

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)離心30秒，倒掉濾出液。

7. 將剩余混合液加到吸附柱中(吸附柱放入收集管中),12000rpm(~13,000g)離心30秒,倒掉濾過液。

8. 向吸附柱CG中加入500 μl 漂洗緩沖液WB1(使用前請先檢查是否已加入無水乙醇),12,000 rpm (~13,000×g )  離心30 秒，倒掉廢液。

9. 向吸附柱CG中加入600 μl 漂洗緩沖液PW(使用前請先檢查是否已加入無水乙醇)，12,000 rpm (~13,000g) 離心30 秒，倒掉廢液，吸附柱CG放入收集管中。

10. 向吸附柱CG中加入600 μl漂洗緩沖液PW，12,000 rpm (~13,000g) 離心30秒，倒掉廢液，將吸附柱CG放入收集管中。

11. 12,000 rpm(~13,000g)離心2分鐘，以徹底晾干吸附材料中殘余的漂洗液。

注：此步驟非常重要，其目的是將吸附柱中殘余的漂洗液去除。漂洗液中乙醇的殘留會影響后續(xù)的酶反應（酶切、PCR等）實驗。

12. 將吸附柱CG轉入一個干凈的1.5ml離心管中，向吸附膜的中間部位懸空滴加50–100 μl經70℃水浴預熱的洗脫緩沖液EB，室溫放置2分鐘，12,000 rpm(~13,000g)離心2分鐘。

注:  = 1 \* GB3 ① 洗脫緩沖液體積最好不少于50 μl，體積過小影響回收效率。

= 2 \* GB3 ② 洗脫液的pH值對于洗脫效率有很大影響。若用水做洗脫液應保證其pH值在7.0-8.5范圍內，pH值低于7.0會降低洗脫效率。

13. DNA產物-20℃保存。

大量操作步驟（針對含微量核酸的樣品）

1. 稱1-5 g土壤置于10ml離心管中，加入1gGlass Beads，再加入3ml 緩沖液R1與200μl 緩沖液R2。渦旋器高速震蕩3-5分鐘。

2. 加入600μl 緩沖液R3，渦旋混勻。70℃水浴處理10分鐘。期間振蕩幾次。

3.3000g離心3分鐘，轉移上清到新的離心管中，加入550μl 緩沖液R4混勻。

4. 冰上放置5分鐘。8000 g離心10分鐘。轉移上清到新的離心管中。

5. 以下按標準操作步驟的第五步繼續(xù)操作。

● DNA濃度及純度檢測：

基因組DNA片段的大小與樣品保存時間、操作過程中的剪切力等因素有關。提取的DNA片段可用瓊脂糖凝膠電泳和紫外分光光度計檢測濃度與純度?？膳渲?.8-1.0％瓊脂糖凝膠，使用λ/HindIII判斷基因組的大小，完整的基因組大小應在23kb以上。使用分光光度計檢測時， OD₂₆₀/OD₂₈₀比值應為1.7–1.9之間，如果洗脫時不使用洗脫緩沖液，而使用去離子水洗脫，比值可能偏低，但并不表明DNA純度不高。

● 常見問題分析：

 實驗示例：

Buffer R2去除腐殖酸效果對比圖
左圖-步驟1提取時使用了Brffer R2，顏色明顯淺很多(左1,左2)，
說明Buffer R2對去除腐殖酸、色素等效果明顯
右圖-步驟3上清加入Buffer R4進一步處理，離心去除雜質，
裂解時加有Buffer R2的，再經Buffer R4處理幾乎不再有顏色(右1,右2)，
而不加Buffer R2的顏色還很深(右3，右4)。

1, 2: SDS高鹽酚氯仿抽提法
3, 4: 土壤基因組DNA提取試劑盒(RTG2403)提取
1, 3: 為花基土壤
2, 4: 河邊淤泥

核酸提取產品發(fā)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 瓊脂糖凝膠回收試劑盒

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瓊脂糖凝膠回收試劑盒

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 瓊脂糖凝膠回收試劑盒

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 瓊脂糖凝膠回收試劑盒

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 瓊脂糖凝膠回收試劑盒

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產物純化試劑盒

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產物純化試劑盒

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 瓊脂糖凝膠回收試劑盒

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產物純化試劑盒

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 普通質粒小提試劑盒

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 普通質粒小提試劑盒

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 動物/細胞基因組DNA提取試劑盒

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 高純質粒小提試劑盒

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 瓊脂糖凝膠回收試劑盒

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 普通質粒小提試劑盒

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 真菌基因組DNA提取試劑盒

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