Insect Genomic DNA Isolation Kit

● 试剂盒内容及保存：

● 储存条件和效期：

室温保存，12个月内有效。缓冲液IL与缓冲液 IB可能有沉淀产生，37℃水浴溶解后即可。RNase A和蛋白酶K常温运输，-20℃保存。

● 产品简介：

该试剂盒采用独特的裂解液能够有效除去多糖多酚等，能够从昆虫、软体动物、节肢动物、蛔虫等样品中提取DNA，保存在醇类的样品也适用于本试剂盒的提取。一次操作可以处理小于50mg组织，样品经裂解液消化，氯仿分离除去大部分的多糖多酚，再经分离柱进一步纯化，便可得到高纯度的DNA。所得的DNA可以用于PCR，Southern杂交，酶切消化等实验。

● 准备工作：

1. 准备65℃水浴；无水乙醇；氯仿；制冰机；1.5ml离心管；2ml离心管

2. 按照标签所示在缓冲液IB中加入异丙醇，在DNA Wash Buffer和缓冲液WB中加入无水乙醇，混匀后盖紧瓶盖后常温贮存备用。

3. 每次使用前请检查缓冲液IL，缓冲液IB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常温下离心。

1. 液氮充分研磨样品。

2. 收集研磨成粉末的50 mg样品，置于1.5ml离心管中。

3. 加入350 μl65℃预热的缓冲液 IL，并加入20μl 蛋白酶K剧烈地漩涡振荡，确保所有的组织团都分散均匀。

4.65℃水浴20-30min。水浴期间颠倒样品数次。

5. 加入350 μl氯仿，充分混匀，12,000 rpm (~13,400×g )  离心5分钟。

6. 小心地把上清液吸至另一新的1.5ml离心管中。注意确保不要打散沉淀团或把组织碎片也一起转移。 

7. 加入4 μl RNase A，涡旋混匀。常温放置2min。

8. 加入等体积的缓冲液 IB（请确保已经按照标签加入异丙醇），充分混匀。如：向300μl上清中加入300 μl 缓冲液 IB。  

9. 把上述混匀的液体转移到吸附柱CG上。10,000×g离心1 min以结合DNA，弃去滤出液体。纯化柱最大容量为750 μl,如果混合液大于750 μl,请分两次过柱。 

10. 将吸附柱重新套回收集管中，加入500μl 缓冲液WB（请确保已经按照标签加入无水乙醇）至柱子中，10,000×g离心1min,倒弃流出液；

11. 将吸附柱重新套回收集管中，加入600μl DNA Wash Buffer（请确保已经按照标签加入无水乙醇）至柱子中，10,000×g离心1min,倒弃流出液；

注意：DNA Wash Buffer使用前须按要求用无水乙醇稀释。如果放入冰箱中，使用前须恢复到室温。 

12.再加入600μl DNA Wash Buffer（请确保已经按照标签加入无水乙醇）至柱子中，8,000×g离心1min,弃去流出液；

13. 将吸附柱重新套回2ml收集管中，最大转速（>13000×g）离心空结合柱1min以干燥柱子的基质；这一步对下面的洗脱步骤至关重要。 

14. 将柱子置于1.5 ml灭菌离心管，加入50-150 μl  65℃预热的洗脱缓冲液EB至柱子的膜中央。常温静置5 min； 

15. 常温下，离心(>13000g)1min，以洗脱DNA。保留含DNA的流出液。将DNA储于-20℃。

● 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。

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