昆蟲(chóng)基因組DNA提取試劑盒

Insect Genomic DNA Isolation Kit

● 試劑盒內(nèi)容及保存：

● 儲(chǔ)存條件和效期：

室溫保存，12個(gè)月內(nèi)有效。緩沖液IL與緩沖液 IB可能有沉淀產(chǎn)生，37℃水浴溶解后即可。RNase A和蛋白酶K常溫運(yùn)輸，-20℃保存。

● 產(chǎn)品簡(jiǎn)介：

該試劑盒采用獨(dú)特的裂解液能夠有效除去多糖多酚等，能夠從昆蟲(chóng)、軟體動(dòng)物、節(jié)肢動(dòng)物、蛔蟲(chóng)等樣品中提取DNA，保存在醇類(lèi)的樣品也適用于本試劑盒的提取。一次操作可以處理小于50mg組織，樣品經(jīng)裂解液消化，氯仿分離除去大部分的多糖多酚，再經(jīng)分離柱進(jìn)一步純化，便可得到高純度的DNA。所得的DNA可以用于PCR，Southern雜交，酶切消化等實(shí)驗(yàn)。

● 準(zhǔn)備工作：

1. 準(zhǔn)備65℃水??；無(wú)水乙醇；氯仿；制冰機(jī)；1.5ml離心管；2ml離心管

2. 按照標(biāo)簽所示在緩沖液IB中加入異丙醇，在DNA Wash Buffer和緩沖液WB中加入無(wú)水乙醇，混勻后蓋緊瓶蓋后常溫貯存?zhèn)溆谩?

3. 每次使用前請(qǐng)檢查緩沖液IL，緩沖液IB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(biāo)準(zhǔn)操作步驟：

如非指出，所有離心步驟均為使用臺(tái)式離心機(jī)在常溫下離心。

1. 液氮充分研磨樣品。

2. 收集研磨成粉末的50 mg樣品，置于1.5ml離心管中。

3. 加入350 μl65℃預(yù)熱的緩沖液 IL，并加入20μl 蛋白酶K劇烈地漩渦振蕩，確保所有的組織團(tuán)都分散均勻。

4.65℃水浴20-30min。水浴期間顛倒樣品數(shù)次。

5. 加入350 μl氯仿，充分混勻，12,000 rpm (~13,400×g )  離心5分鐘。

6. 小心地把上清液吸至另一新的1.5ml離心管中。注意確保不要打散沉淀團(tuán)或把組織碎片也一起轉(zhuǎn)移。 

7. 加入4 μl RNase A，渦旋混勻。常溫放置2min。

8. 加入等體積的緩沖液 IB（請(qǐng)確保已經(jīng)按照標(biāo)簽加入異丙醇），充分混勻。如：向300μl上清中加入300 μl 緩沖液 IB。  

9. 把上述混勻的液體轉(zhuǎn)移到吸附柱CG上。10,000×g離心1 min以結(jié)合DNA，棄去濾出液體。純化柱最大容量為750 μl,如果混合液大于750 μl,請(qǐng)分兩次過(guò)柱。 

10. 將吸附柱重新套回收集管中，加入500μl 緩沖液WB（請(qǐng)確保已經(jīng)按照標(biāo)簽加入無(wú)水乙醇）至柱子中，10,000×g離心1min,倒棄流出液；

11. 將吸附柱重新套回收集管中，加入600μl DNA Wash Buffer（請(qǐng)確保已經(jīng)按照標(biāo)簽加入無(wú)水乙醇）至柱子中，10,000×g離心1min,倒棄流出液；

注意：DNA Wash Buffer使用前須按要求用無(wú)水乙醇稀釋。如果放入冰箱中，使用前須恢復(fù)到室溫。 

12.再加入600 μl DNA Wash Buffer（請(qǐng)確保已經(jīng)按照標(biāo)簽加入無(wú)水乙醇）至柱子中，8,000×g離心1min,棄去流出液；

13. 將吸附柱重新套回2ml收集管中，最大轉(zhuǎn)速（>13000×g）離心空結(jié)合柱1min以干燥柱子的基質(zhì)；這一步對(duì)下面的洗脫步驟至關(guān)重要。 

14. 將柱子置于1.5 ml滅菌離心管，加入50-150 μl  65℃預(yù)熱的洗脫緩沖液EB至柱子的膜中央。常溫靜置5 min； 

15. 常溫下，離心(>13000g)1min，以洗脫DNA。保留含DNA的流出液。將DNA儲(chǔ)于-20℃。

● DNA濃度及純度檢測(cè)：

基因組DNA片段的大小與樣品保存時(shí)間、操作過(guò)程中的剪切力等因素有關(guān)。提取的DNA片段可用瓊脂糖凝膠電泳和紫外分光光度計(jì)檢測(cè)濃度與純度。可配制0.8-1.0％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(yīng)在23 kb以上。使用分光光度計(jì)檢測(cè)時(shí)， OD₂₆₀/OD₂₈₀比值應(yīng)為1.7–1.9之間，如果洗脫時(shí)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。

核酸提取產(chǎn)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(chǎn)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(chǎn)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(chǎn)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(zhì)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(zhì)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(dòng)物/細(xì)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(zhì)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(zhì)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