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- Tateno Minako
- Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, Laboratory for Genome Exploration Research Project, GSC, Genome Science Laboratory, RIKEN
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- Hayashizaki Yoshihide
- Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, Laboratory for Genome Exploration Research Project, GSC, Genome Science Laboratory, RIKEN Institute of Basic Medical Science, University of Tsukuba
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抄録
We describe the newest version of the protocol to construct a bacterial artificial chromosome (BAC) library which carries uniform and larger insert DNA. This protocol is based on the five major improvements reported by Osoegawa et al. (1998) to increase the cloning efficiency and decrease the level of non-insert clones. The first improvement is a purification of high molecular weight (HMW) DNA with a pulse field gel electrophoresis, which eliminates inhibitory contaminants for restriction enzymes and leads to the increase of digestion efficiency of HMW DNA. The second is the doublesize fractionation method, in which partially digested DNA is subjected to an electrophoresis in reverse direction to remove smaller DNA fragments, and then size-fractionated in forward direction. This step enables the preparation of uniform and large-size insert DNA without contamination by smaller DNA. The third is recovering the size-fractionated DNA from agarose gel by the use of electroelution, rather than the use of β agarase as in conventional methods. This step dramatically increases both the purity and integrity of large DNA fragments, leading to an increase of cloning efficiency. The fourth step is the concentration of the ligation products using dialysis against polyethylene glycol buffer with high osmotic pressure. This step increases the number of the transformants produced in a single transformation trial, resulting in saving time and cost. The fifth is the most effective improvement in preparing a BAC vector to minimize a level of non-insert clones. This step employs the ligase-treatment of vector fraction to completely remove the molecules with phosphorylated ends.<br> In this paper, we introduce the precise optimal conditions in each step, which enabled us to make BAC libraries with around 200 kb insert DNA. One example is the RPCI23 library constructed from C57BL/6J female mice. This library consists of approximately 180,000 clones with an average insert size of 197 kb and contains only 6.8 % noninsert clones. This library covers the mouse genome 11.2 times with the most uniform and largest insert DNA except for YAC libraries.
収録刊行物
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- bioimages
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bioimages 6 (3-4), 117-125, 1998
日本バイオイメージング学会
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詳細情報
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- CRID
- 1390564227319187072
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- NII論文ID
- 10002038657
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- NII書誌ID
- AA11084187
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- ISSN
- 09192719
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- CiNii Articles
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- 抄録ライセンスフラグ
- 使用不可