10bp DNA ladder（10-100 bp）

本産品是由8條帶狀雙鏈DNA條帶組成的精準定量Marker，适用于聚丙烯酰胺凝膠電(diàn)泳中DNA條帶大小(xiǎo)和含量的分析。8條帶的大小(xiǎo)分别為(wèi)10，15，20，25，30，40，50，100bp。其中25 bp條帶為(wèi)加亮帶，含量為(wèi)100 ng/5 μl，其餘條帶濃度為(wèi)50 ng/5 μl。

本産品為(wèi)雙鏈DNA Marker，适用于非變性的PAGE電(diàn)泳和瓊脂糖凝膠電(diàn)泳，不适用于尿素PAGE電(diàn)泳。由于片段很小(xiǎo)，不建議用瓊脂糖凝膠電(diàn)泳分離。

按照(zhào)每次上(shàng)樣5 μl計算(suàn)，該産品可以使用50次。

● 儲存、效期及運輸條件(jiàn)：

-20℃ 貯存;有效期3年(nián)；濕冰運輸。

● 使用方法：

一(yī)、 TBE-PAGE凝膠分離：

1、制備凝膠步驟：

1.1參照(zhào)凝膠模具說明書，裝配好凝膠模具。

1.2按照(zhào)表一(yī)将不同體積的成分在小(xiǎo)燒杯或試管中混合；最後加入10%APS和TEMED，輕輕攪拌使其混勻，避免産生(shēng)氣泡。

注：10 bp DNA ladder 适用于配制20%TBE-PAGE膠。

1.3在凝膠模具中灌入适量分離膠溶液（對于mini-gel，凝膠液加至約距前玻璃闆頂端1.5 cm或距梳齒約0.5 cm即可），然後在分離膠溶液上(shàng)輕輕覆蓋一(yī)層1-2 cm的無水(shuǐ)乙醇，使凝膠表面保持平整。

1.4靜(jìng)置10-20分鍾，待分離膠和乙醇層之間出現一(yī)個(gè)清晰的界面後，說明凝膠已聚合

表一(yī) TBE-PAGE分離膠配方表 (總體積5 ml，适用于1mm厚度小(xiǎo)闆膠)

		各組份體積（ml）
最佳DNA分離範圍	凝膠濃度	滅菌水(shuǐ)	30%PAA（29：1）	5×TBE	10%APS	TEMED
70-450 bp	6%	3	1.0	1.0	0.05	0.005
60-460 bp	8%	2.66	1.34	1.0	0.05	0.005
50-300 bp	10%	2.33	1.67	1.0	0.05	0.005
40-200 bp	12%	2	2.0	1.0	0.05	0.005
25-150 bp	15%	0.5	2.5	1.0	0.05	0.005
5-100 bp	20%	0.66	3.34	1.0	0.05	0.005

1.5去除覆蓋在分離膠上(shàng)的乙醇；按照(zhào)表二将不同體積成分在一(yī)個(gè)小(xiǎo)燒杯或試管中混合；最後加入10%過硫酸铵和TEMED，輕輕攪拌使其混勻，避免産生(shēng)氣泡。

表二 TBE-PAGE 4%濃縮配方表 (總體積1.5 ml，适用于1mm厚度小(xiǎo)闆膠)

	各組份體積（ml）
凝膠濃度	滅菌水(shuǐ)	30%PAA（29：1）	5×TBE	10%APS	TEMED
4%	1.0	0.2	0.3	0.02	0.002

1.6将濃縮膠溶液加至分離膠的上(shàng)面，直至凝膠溶液到(dào)達前玻璃闆的頂端；将梳子插入凝膠内，避免産生(shēng)氣泡。

1.7靜(jìng)置30-60分鍾，等待濃縮膠聚合。

注：凝膠的聚合時間與環境溫度有關。夏天溫度較高(gāo)時，聚合較快；冬天氣溫低(dī)時，聚合時間會(huì)延長(cháng)。可以根據環境溫度的不同調節APS的加入量。

2、電(diàn)泳：

2.1 将凝膠闆固定在電(diàn)泳裝置上(shàng)，往上(shàng)槽和下(xià)槽中加入1×TBE電(diàn)泳液，1 ml吸頭沖洗加樣孔1-2次。

2.2 取待測樣品，加入相(xiàng)應體積6×Native PAGE DNA上(shàng)樣緩沖液，如5 μl樣品加1 μl 上(shàng)樣緩沖液，短暫離心後取5-10 μl上(shàng)樣。 10 bp DNA ladder 1 mm厚10齒梳子上(shàng)樣5 μl，其餘梳齒和厚度凝膠适量調整上(shàng)樣量。

2.3 連接電(diàn)源線，打開(kāi)電(diàn)源開(kāi)關。200 V穩壓電(diàn)泳，至溴酚藍指示前沿距離玻璃下(xià)沿1 cm時結束電(diàn)泳。

TBE-PAGE電(diàn)泳
恒電(diàn)壓	起始電(diàn)流	結束電(diàn)流	電(diàn)泳時間
200 V	20-25 mA/闆膠	10-15 mA/闆膠	50+min

20%T3.3C TBE-PAGE

3、染色：

3.1 漂洗：玻璃闆上(shàng)拆下(xià)凝膠後，适量蒸餾水(shuǐ)漂洗3-5分鍾。

3.2 染色液配制：

TBE-PAGE膠可以使用EB或RealSafe類染料後染染色，以下(xià)程序用RealSafe Red核酸染料（貨号：GR002）進行染色。即用型染色液配制：

	即用型RealSafe核酸染色液
	100 ml
1×TBE	100 ml
RealSafe Red核酸染料	20 μl

3.3染色和觀察：

凝膠浸泡于即用型染色液中，常溫避光(guāng)搖床40-60 rpm染色30 分鍾。紫外光(guāng)下(xià)觀察。

4、實驗示例：

20％TBE-PAGE電(diàn)泳

左1-7：10,15,20,25,30,40,50 bp

右1：10 bp DNA ladder

凝膠長(cháng)度8 cm

電(diàn)壓200 V 起始電(diàn)流25 mA，終止電(diàn)流13 mA

電(diàn)泳緩沖液：1×TBE

電(diàn)泳時間：50分鍾

染色：RealSafe Red核酸染料後染30分鍾

使用DNA Marker産品發表部分文章列表：

1. [2021 IF=1.17] Dendrostoma covidicola sp. nov. (Erythrogloeaceae, Diaporthales) on Fagus sylvatica from Sichuan Province, China.

Author: MILAN C. SAMARAKOON, ITTHAYAKORN PROMPUTTHA & JIAN-KUI (JACK) LI

Marker: RTM415，D2000 DNA ladder

Journal: Phytotaxa. 2021，483 (2): 085–094.

Institution：University of Electronic Science and Technology of China

Paper link：https://doi.org/10.11646/phytotaxa.483.2.1

2.[2021 IF=6.057] MUC1 detection and in situ imaging method based on aptamer conformational switch and hybridization chain reaction.

Marker :RTM403，DNA Marker III

Author: ZihanSong，ShengCai.

Journal: Talanta. Vol 239, 2022, 123129

Institution： Zhejiang University

Paper link：https://doi.org/10.1016/j.ijbiomac.2021.11.194

3. [2021 IF=3.24] Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility.

Marker :RTM404，DNA Marker IV

Author: Xiankai Liu, Dongshu Wang, Huagui Wang, Erling Feng, Li Zhu, Hengliang Wang

Journal: PLos one，January 2012 | Volume 7 | Issue 1 | e29875

Institution：State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology

Paper link： https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029875

4. [2021 IF=3.24] Identification of a Novel Fungus, Leptosphaerulina chartarum SJTU59 and Characterization of Its Xylanolytic Enzymes.

Marker :RTM409，100bp DNA ladder

Author: Qiong Wu, Yaqian Li, Yingying Li, Shigang Gao, Meng Wang, Tailong Zhang, Jie Chen

Journal: PLos one，September 2013 | Volume 8 | Issue 9 | e737

Institution：School of Agriculture and Biology, Shanghai Jiao Tong University

Paper link：https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073729

5. [2021 IF=3.08] Low genetic diversity and local adaptive divergence of Dracaena cambodiana (Liliaceae) populations associated with historical population bottlenecks and natural selection: an endangered long-lived tree endemic to Hainan Island, China.

Marker :RTM409，100bp DNA ladder

Author: D.-J. Zheng, L.-S. Xie, J.-H. Zhu，Z.-L. Zhang

Journal: Plant Biology，14 (2012) 828–838

Institution：Key Laboratory of Crop Genetics and Breeding of Hainan Province, Hainan Academy of Agricultural Sciences

Paper link：https://onlinelibrary.wiley.com/doi/10.1111/j.1438-8677.2011.00559.x

6. [2021 IF=3.24] Envelope Proteins ofWhite Spot Syndrome Virus (WSSV) Interact with Litopenaeus vannamei Peritrophin-Like Protein (LvPT).

Marker :RTM415，D2000 DNA ladder

Author: Shijun Xie, Xiaojun Zhang, Jiquan Zhang, Fuhua Li, Jianhai Xiang

Journal: Plos One，Published: December 21, 2015

Institution：Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences

Paper link： https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144922

7. [2021 IF=1.17] Rhopalostroma brevistipitatum sp. nov. from Thailand with an extended generic description for Rhopalostroma.

Marker :RTM415，D2000 DNA ladder

Author: DINUSHANI A. DARANAGAMA, XINGZHONG LIU, SUNITA CHAMYUANG, MARC STADLER，ALI H.BAHKALI, KEVIN D. HYDE

Journal: Phytotaxa，2015，227 (3): 229–242

Institution：State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences

Paper link：https://www.biotaxa.org/Phytotaxa/article/view/phytotaxa.227.3.2/0

8. [2021 IF=20.37] A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis)

Marker :RTM415，D2000 DNA ladder

Author: Dhanushka Udayanga & Xingzhong Liu &Pedro W. Crous & Eric H. C. McKenzie &Ekachai Chukeatirote & Kevin D. Hyde

Journal: Fungal Diversity (2012) 56:157–171

Institution：State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences

Paper link：https://link.springer.com/article/10.1007/s13225-012-0190-9

9. [2021 IF=2.9] An easily-performed high-throughput method for plant genomic DNA extraction.

Marker :RTM421，λ DNA/HindIII

Author: Deshui Yu, Ju Zhang, Guangxuan Tan, Ningshu Yu, Qiuyue Wang, Qiqi Duan, Xin

Qi, Mingjiao Cheng, Chunxue Yan, Zhangkun Wei, Zhenmiao Yu, Wenchao Huang, Chengwei Li

Journal: Analytical Biochemistry Volume 569, 15 March 2019, Pages 28-30

Institution：Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal

University

Paper link：

https://www.sciencedirect.com/science/article/abs/pii/S0003269718310637?via%3Dihub

10. [2021 IF=2.88] Genetic diversity and genetic structure of Decalobanthus boisianus in Hainan Island, China.

Marker :RTM415，D2000 DNA ladder

Author: Huan Jiang，Wenxing Long， Hui Zhang，Chengneng Mi，Tao Zhou，Zongzhu Chen

Journal: Ecology and Evolotion Volume 569, 15 March 2019, Pages 28-30

Institution：Institute of Tropical Agriculture and Forestry, Hainan University

Paper link： https://doi.org/10.1002/ece3.5127

11. [2021 IF=5.64] Identi?cation of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata.

Marker :RTM406，DNA Marker VI

Author: Huan Li, Ruiling Bian, Qian Liu, Liu Yang, Tianxing Pang, Lakha Salaipeth,

Ida Bagus Andika, Hideki Kondo and Liying Sun

Journal: Frontiers in Microbiology May 2019 | Volume 10 | Article 1076

Institution：State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University

Paper link： https://www.frontiersin.org/articles/10.3389/fmicb.2019.01076/full

12. [2021 IF=2.6] Natural occurrence of Alternaria mycotoxins in wheat and potential of reducing associated risks using magnolol.

Marker :RTM415，D2000 DNA ladder

Author: Dongmei Jiang, Dizhe Wei, Hongtao Li, Liuqing Wang, Nan Jiang,Yunchao Li and Meng Wang

Journal: Journal of the Science of Food and Agriculture Volume101, Issue7，2021，3071-3077

Institution：Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Sciences

Paper link：https://onlinelibrary.wiley.com/doi/10.1002/jsfa.10901

13. [2021 IF=20.37] Taxonomy, Phylogeny, Molecular Dating and Ancestral State Reconstruction of Xylariomycetidae (Sordariomycetes)

Marker :RTM415，D2000 DNA ladder

Author: Kevin D Hyde，Jian-Kui Liu

Journal: Fungal Diversity volume 112, pages 1–88 (2022)

Institution：School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China

Paper link：https://link.springer.com/article/10.1007/s13225-021-00495-5

14. [2021 IF=8.025] A method for high-concentration agarose gel preparation and its

application in high-resolution separation of low-molecular-weight nucleic acids and proteins.

Marker :RTM443，10bp DNA ladder

Author: Lili Chang , Dan Wang, Cunzhi Peng, Qi Wang, Bingqiang Xu, Zheng Tong

Journal: International Journal of Biological Macromolecules 231 (2023) 123358

Institution：Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural

Sciences

Paper link：https://doi.org/10.1016/j.ijbiomac.2023.123358

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