菖蒲芋螺不同大小基因组DNA片段的分离制备

丁青; 刘月鹏; 朱晓鹏; 吴科榜; 罗素兰; 长孙东亭

doi:10.15886/j.cnki.rdswxb.2017.03.004

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菖蒲芋螺不同大小基因组DNA片段的分离制备

丁青, 刘月鹏, 朱晓鹏, 吴科榜, 罗素兰, 长孙东亭

文章导航 > 热带生物学报（中英文） > 2017 > 8(3): 267-276

丁青, 刘月鹏, 朱晓鹏, 吴科榜, 罗素兰, 长孙东亭. 菖蒲芋螺不同大小基因组DNA片段的分离制备[J]. 热带生物学报（中英文）, 2017, 8(3): 267-276. doi: 10.15886/j.cnki.rdswxb.2017.03.004

引用本文:

DING Qing, LIU Yuepeng, ZHU Xiaopeng, WU Kebang, LUO Sulan, ZHANGSUN Dongting. Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum[J]. Journal of Tropical Biology, 2017, 8(3): 267-276. doi: 10.15886/j.cnki.rdswxb.2017.03.004

Citation:

DING Qing, LIU Yuepeng, ZHU Xiaopeng, WU Kebang, LUO Sulan, ZHANGSUN Dongting. Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum[J]. Journal of Tropical Biology, 2017, 8(3): 267-276. doi: 10.15886/j.cnki.rdswxb.2017.03.004

菖蒲芋螺不同大小基因组DNA片段的分离制备

DOI: 10.15886/j.cnki.rdswxb.2017.03.004

1. 海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228;
2. 海南大学热带农林学院, 海口 570228

基金项目:

长江学者和创新团队发展计划(IRT-15R15)

国家自然科学基金(41366002)

详细信息

第一作者:
丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

通信作者:
长孙东亭(1964-),男,研究员.研究方向:生物技术与海洋药物.E-mail:zhangsundt@163.com

中图分类号: Q33

Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum

1. Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drug of Haikou, Hainan University, Haikou 570228, China;
2. College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

摘要: 使用常规酚/氯仿抽提法(CTAB法)、离心柱法和磁珠吸附法,分别从菖蒲芋螺毒腺、肌肉及肝胰脏中提取基因组DNA,利用普通琼脂糖凝胶电泳与脉冲场电泳(PFGE)对基因组DNA片段进行分离,并检测提取DNA的大小和分布范围,同时,对脉冲场电泳条件进行了优化。结果表明,3种方法均可提取基因组DNA,其中CTAB法和离心柱法提取的毒腺DNA纯度高,产率大,可用于后续实验,而磁珠法产率适中且纯度不高,但离心柱法的DNA片段较小,大多在9 kb以下,而CTAB法提取的DNA片段较大,获得的20 kb以上的大片段量较多,更适合于大片段基因组文库的构建。3种芋螺组织中,肝胰脏产率最高但片段弥散有降解,毒腺DNA片段较大且集中,产率也较高,而肌肉的DNA片段产率最低且片段较小。因此,用CTAB法提取菖蒲芋螺毒腺的DNA更适合构建大片段基因组DNA文库。筛选高质量的菖蒲芋螺的DNA,利用优化的脉冲场电泳条件进行分离回收,获得了不同大小片段的DNA,为后续菖蒲芋螺不同载体系统的基因组文库构建奠定了基础。
- 菖蒲芋螺 /
- 基因组DNA提取方法 /
- 基因组DNA片段 /
- 分离制备 /
- 脉冲场电泳
Abstract: In order to provide basis for construction of the genomic library of Conus vexillum（C.vexillum）,different methods were used for isolation and extraction of different size fragments of genomic DNA from C.vexillum native to Hainan.Genomic DNA was isolated from venom glands,hepatopancreas and muscle of C.vexillum using three methods of CTAB,spin column and magnetic bead respectively.The genomic DNA fragments were separated by agarose gel electrophoresis and pulsed-field gel electrophoresis,of which the size and distribution range of extracted DNA was detected.The results showed that the genomic DNA could be extracted by all the three methods.The purity and yield of DNAs isolated by CTAB and centrifugal column methods using venom gland of C.vexillum were good and high,which could meet the following experimental requirements.While the yield of the magnetic bead method is less and the purity is not good.The length of most DNA fragments isolated by the centrifugal column method was less than 9 kb,while most fragments’ sizes isolated by CTAB method were more than 20 kb.Among the three kinds of organs,hepatopancreas had highest yield of DNA,but its fragment was dispersed with degradation happened,the DNA yield of venom glands was high but less than hepatopancreas slightly,its integrity was the best;however the DNA yield of muscle was lowest and its fragments was smaller.The high quality genomic DNA fragments with different sizes were separated and prepared successfully by optimized pulsed-field gel electrophoresis.In conclusion,using CTAB method to isolate C.vexillum DNA is better than the other methods for big fragment genomic DNA library construction.Different size genomic DNA fragments recovered by pulsed-field gel electrophoresis would lay a foundation for further genomic library construction of C.vexillum using different gene library vector systems.
- Conus vexillum native to Hainan /
- Genomic DNA extraction method /
- Different size fragments of genomic DNA /
- Isolation and preparation /
- Pulsed field electrophoresis

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菖蒲芋螺不同大小基因组DNA片段的分离制备

DOI: 10.15886/j.cnki.rdswxb.2017.03.004

1. 海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228;

2. 海南大学热带农林学院, 海口 570228

基金项目:

长江学者和创新团队发展计划(IRT-15R15)

国家自然科学基金(41366002)

作者简介:
丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

通讯作者: 长孙东亭(1964-),男,研究员.研究方向:生物技术与海洋药物.E-mail:zhangsundt@163.com

收稿日期: 2017-03-13
修回日期: 2017-04-17

中图分类号: Q33

关键词:

摘要: 使用常规酚/氯仿抽提法(CTAB法)、离心柱法和磁珠吸附法,分别从菖蒲芋螺毒腺、肌肉及肝胰脏中提取基因组DNA,利用普通琼脂糖凝胶电泳与脉冲场电泳(PFGE)对基因组DNA片段进行分离,并检测提取DNA的大小和分布范围,同时,对脉冲场电泳条件进行了优化。结果表明,3种方法均可提取基因组DNA,其中CTAB法和离心柱法提取的毒腺DNA纯度高,产率大,可用于后续实验,而磁珠法产率适中且纯度不高,但离心柱法的DNA片段较小,大多在9 kb以下,而CTAB法提取的DNA片段较大,获得的20 kb以上的大片段量较多,更适合于大片段基因组文库的构建。3种芋螺组织中,肝胰脏产率最高但片段弥散有降解,毒腺DNA片段较大且集中,产率也较高,而肌肉的DNA片段产率最低且片段较小。因此,用CTAB法提取菖蒲芋螺毒腺的DNA更适合构建大片段基因组DNA文库。筛选高质量的菖蒲芋螺的DNA,利用优化的脉冲场电泳条件进行分离回收,获得了不同大小片段的DNA,为后续菖蒲芋螺不同载体系统的基因组文库构建奠定了基础。

English Abstract

Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum

1. Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drug of Haikou, Hainan University, Haikou 570228, China;

2. College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

Received Date: 2017-03-13
Rev Recd Date: 2017-04-17

Keywords:

Abstract: In order to provide basis for construction of the genomic library of Conus vexillum（C.vexillum）,different methods were used for isolation and extraction of different size fragments of genomic DNA from C.vexillum native to Hainan.Genomic DNA was isolated from venom glands,hepatopancreas and muscle of C.vexillum using three methods of CTAB,spin column and magnetic bead respectively.The genomic DNA fragments were separated by agarose gel electrophoresis and pulsed-field gel electrophoresis,of which the size and distribution range of extracted DNA was detected.The results showed that the genomic DNA could be extracted by all the three methods.The purity and yield of DNAs isolated by CTAB and centrifugal column methods using venom gland of C.vexillum were good and high,which could meet the following experimental requirements.While the yield of the magnetic bead method is less and the purity is not good.The length of most DNA fragments isolated by the centrifugal column method was less than 9 kb,while most fragments’ sizes isolated by CTAB method were more than 20 kb.Among the three kinds of organs,hepatopancreas had highest yield of DNA,but its fragment was dispersed with degradation happened,the DNA yield of venom glands was high but less than hepatopancreas slightly,its integrity was the best;however the DNA yield of muscle was lowest and its fragments was smaller.The high quality genomic DNA fragments with different sizes were separated and prepared successfully by optimized pulsed-field gel electrophoresis.In conclusion,using CTAB method to isolate C.vexillum DNA is better than the other methods for big fragment genomic DNA library construction.Different size genomic DNA fragments recovered by pulsed-field gel electrophoresis would lay a foundation for further genomic library construction of C.vexillum using different gene library vector systems.

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菖蒲芋螺不同大小基因组DNA片段的分离制备

DOI: 10.15886/j.cnki.rdswxb.2017.03.004

第一作者: 丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

通信作者: 长孙东亭(1964-),男,研究员.研究方向:生物技术与海洋药物.E-mail:zhangsundt@163.com

Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum

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出版历程

菖蒲芋螺不同大小基因组DNA片段的分离制备

DOI: 10.15886/j.cnki.rdswxb.2017.03.004

1. 海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228; 2. 海南大学热带农林学院, 海口 570228

作者简介: 丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

通讯作者: 长孙东亭(1964-),男,研究员.研究方向:生物技术与海洋药物.E-mail:zhangsundt@163.com

English Abstract

Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum

1. Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drug of Haikou, Hainan University, Haikou 570228, China; 2. College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

全文HTML

目录

喜报！《热带生物学报（中英文）》影响因子稳步攀升，再创历史新高！

第一作者:
丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

通信作者:
长孙东亭(1964-),男,研究员.研究方向:生物技术与海洋药物.E-mail:zhangsundt@163.com

1. 海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228;

2. 海南大学热带农林学院, 海口 570228

作者简介:
丁青(1991-),女,海南大学热带农林学院2014级硕士研究生.E-mail:598594694@qq.com

1. Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drug of Haikou, Hainan University, Haikou 570228, China;

2. College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China