全血基因組DNA小量提取試劑盒

Total DLood DNA Miniprep Kit

● 試劑盒內(nèi)容及保存：

● 儲(chǔ)存條件和效期：

該試劑盒常溫貯存，常溫運(yùn)輸。開(kāi)封后有效期一年。

● 產(chǎn)品簡(jiǎn)介：

全血基因組DNA 小量提取試劑盒可從200-400 μl體積新鮮、冷凍或抗凝全血樣本中快速分離純化基因組DNA。該試劑盒可以在30分鐘內(nèi)完成單個(gè)或多個(gè)樣品的操作。 提取過(guò)程無(wú)需使用蛋白酶K消化，不需要酚氯仿抽提， 也無(wú)需耗時(shí)的異丙醇沉淀等過(guò)程 。

使用該試劑盒提取到的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)備無(wú)水乙醇；制冰機(jī)；1.5ml離心管；2ml離心管；離心機(jī)

2. 按照標(biāo)簽所示在去蛋白液GD和漂洗液PW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DL1和DL2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。

注意：以下方案適用于400 μl體積新鮮或冷凍血液樣本。如果使用低于400 μl的全血樣本，必須按比例減少緩沖液DL1和DL2的用量或者用1×PBS溶液補(bǔ)齊至400 μl，嚴(yán)格按照緩沖液DL1：全血：緩沖液DL2=3：4：3體積比進(jìn)行操作，否則將導(dǎo)致后續(xù)操作無(wú)法進(jìn)行。

1. 加入300μl緩沖液DL1于1.5 ml離心管中。

2. 加入400 μl抗凝全血，漩渦劇烈震蕩30秒。 注：如果低于400 μl血液，用1×PBS補(bǔ)齊到400 μl。

3. 加入300μl緩沖液DL2，劇烈搖動(dòng)離心管3-5次，漩渦劇烈震蕩30秒-60秒。

注：加入緩沖液DL2后，會(huì)出現(xiàn)大量血紅蛋白沉淀，必須劇烈震蕩。

4. 12000 rpm 離心2分鐘。

5. 把吸附柱裝在2ml收集管中（已提供），將第4步得到的上清溶液全部轉(zhuǎn)入柱中（小心別觸及沉淀），12000 rpm離心1min，倒棄流出液重新套上收集管。

注：吸附柱的承受最大體積是700 μl，如上清超過(guò)此體積，可分2次離心，保證所有上清都加到柱中。

6.加入500 μl 去蛋白液GB（注意是否已經(jīng)加入乙醇）至柱子中，12000 rpm離心1min，棄去流出液。

注：吸附柱膜上會(huì)有血色素殘余，這是正常現(xiàn)象，可以被去蛋白液GD和漂洗液PW洗去。

7. 將吸附柱重新套回2ml收集管中，加入700μl 漂洗液PW（注意是否已經(jīng)加入乙醇）至柱子中，12000 rpm離心1min,倒棄流出液；

8. 再加入500μl 漂洗液PW至柱子中，12000 rpm離心1min, 棄去流出液；

9. 將吸附柱重新套回2ml收集管中，12000 rpm離心空結(jié)合柱2 min以干燥柱子的基質(zhì)；

注：這一步驟至關(guān)重要，不要省略。 否則殘余乙醇會(huì)影響后續(xù)酶切或PCR實(shí)驗(yàn)。

10. 將柱子置于1.5ml滅菌離心管，加入50-80 μl65℃預(yù)熱的洗脫緩沖液EB或滅菌去離子水至柱子的膜中央。 常溫靜置2 min； 

注： ① 洗脫緩沖液體積最好不少于50 μl，體積過(guò)小影響回收效率。

 ② 洗脫液的pH值對(duì)于洗脫效率有很大影響。若用水做洗脫液應(yīng)保證其pH值在7.0-8.5范圍內(nèi)，pH值低于7.0會(huì)降低洗脫效率。

11. 12000 rpm離心 2 min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?？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(yīng)在23kb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。

● 常見(jiàn)問(wèn)題分析：

 實(shí)驗(yàn)示例：

400μl human blood gDNA 上樣5μl

人血液基因組擴(kuò)增人β-球蛋白 1.3kb 片段

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