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：
|
試劑盒組成
|
RTG2406
（50次）
|
貯存方式
|
|
緩沖液DL1
|
16
ml
|
常溫
|
|
緩沖液DL2
|
16
ml
|
常溫
|
|
去蛋白液GD（濃縮液）
|
30 ml
|
常溫
|
|
漂洗液PW（濃縮液）
|
25
ml
|
常溫
|
|
洗脫緩沖液EB
|
15
ml
|
常溫
|
|
吸附柱CG
|
50個
|
常溫
|
|
收集管（2
ml）
|
50個
|
常溫
|
|
說明書
|
1份
|
|
● 儲存條件和效期：
該試劑盒常溫貯存，常溫運輸。開封后有效期一年。
● 產(chǎn)品簡介：
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)完成單個或多個樣品的操作。 提取過程無需使用蛋白酶K消化，不需要酚氯仿抽提，
也無需耗時的異丙醇沉淀等過程 。
使用該試劑盒提取到的DNA
可以用于PCR，Southern雜交，酶切消化等實驗。
● 準備工作：
1. 準備無水乙醇；制冰機；1.5ml離心管；2ml離心管；離心機
2. 按照標簽所示在去蛋白液GD和漂洗液PW中加入無水乙醇，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溶液補齊至400 μl，嚴格按照緩沖液DL1：全血：緩沖液DL2=3：4：3體積比進行操作，否則將導致后續(xù)操作無法進行。
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補齊到400
μl。
3. 加入300μl緩沖液DL2，劇烈搖動離心管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：吸附柱膜上會有血色素殘余，這是正?，F(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。 否則殘余乙醇會影響后續(xù)酶切或PCR實驗。
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，體積過小影響回收效率。
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(yīng)保證其pH值在7.0-8.5范圍內(nèi)，pH值低于7.0會降低洗脫效率。
11. 12000 rpm離心
2 min洗脫DNA。保留含DNA的流出液。將DNA儲于-20℃。
● 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(guān)。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(yīng)在23kb以上。使用分光光度計檢測時， OD260/OD280比值應(yīng)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：
|
常見問題
|
可能原因
|
建議
|
|
堵柱子
|
樣本體積不對，裂解不完全
|
未按照說明書使用指定體積操作，請嚴格按照緩沖液DL1：全血：緩沖液DL2=3：4：3體積比進行操作
|
|
回收不到DNA或得率低
|
堵柱子
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見上
|
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化療病人全血中提取DNA
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化療病人血液中白細胞數(shù)量減少，導致DNA數(shù)量降低
|
|
全血樣品保存不當，導致全血溶血導致DNA降解
|
4℃冰箱中貯存2周的全血開始出現(xiàn)溶血現(xiàn)象，此時DNA隨貯存時間延長會程序分解，最終導致分離不到電泳可見的DNA；全血應(yīng)該-20℃或-80℃貯存。
|
|
去蛋白液GD和漂洗液PW沒有按說明書加入乙醇稀釋
|
使用前按說明書加入適量的無水乙醇進行稀釋
|
|
DNA純度差
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高A260/A280比率接近2.0
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考慮全血樣品的新鮮程度。陳舊全血中的DNA會降解成小片段DNA或核苷酸混合物，導致A260讀數(shù)變大
|
|
DNA中有RNA污染
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使用非常新鮮的全血提取DNA時電泳結(jié)果會有RNA污染，RNA不能作為擴增模板，不會影響PCR擴增。如要去除RNA，可在步驟1加入緩沖液DL1時同步加入5 μl RNaseA溶液（10mg/ml）
|
|
洗滌時柱中有帶顏色的遺留物
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未按照說明書使用指定體積操作
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請嚴格按照緩沖液DL1：全血：緩沖液DL2=3：4：3體積比進行操作
|
|
去蛋白液GB和漂洗液PW沒有按說明書加入乙醇稀釋
|
使用前按說明書加入適量的無水乙醇進行稀釋
|
實驗示例：
400μl human blood gDNA 上樣5μl
人血液基因組擴增人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(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