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A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the beta-galactosidase (beta-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95 degrees C, 60 degrees C and 72 degrees C) reactions of real-time PCR to detect the beta-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. 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Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution
http://hdl.handle.net/10091/974
http://hdl.handle.net/10091/974da13bd6d-2898-4190-b539-b06f706b96a0
名前 / ファイル | ライセンス | アクション |
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Item type | 学術雑誌論文 / Journal Article(1) | |||||
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公開日 | 2008-02-05 | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源 | http://purl.org/coar/resource_type/c_6501 | |||||
タイプ | journal article | |||||
著者 |
Sakurai, T
× Sakurai, T× Kamiyoshi, A× Watanabe, S× Sato, M× Shindo, T |
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信州大学研究者総覧へのリンク | ||||||
氏名 | Shindo, T | |||||
URL | http://soar-rd.shinshu-u.ac.jp/profile/ja.OCLpOayV.html | |||||
出版者 | ||||||
出版者 | SPRINGER | |||||
引用 | ||||||
内容記述タイプ | Other | |||||
内容記述 | TRANSGENIC RESEARCH. 17(1): 149-155 (2008) | |||||
書誌情報 |
Transgenic research 巻 17, 号 1, p. 149-155, 発行日 2008-03 |
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内容記述 | ||||||
内容記述タイプ | Other | |||||
内容記述 | The original publication is available at www.springerlink.com. | |||||
資源タイプ(コンテンツの種類) | ||||||
内容記述タイプ | Other | |||||
内容記述 | Article | |||||
ISSN | ||||||
収録物識別子タイプ | PISSN | |||||
収録物識別子 | 0962-8819 | |||||
書誌レコードID | ||||||
収録物識別子タイプ | NCID | |||||
収録物識別子 | AA10873487 | |||||
DOI | ||||||
識別子タイプ | DOI | |||||
関連識別子 | https://doi.org/10.1007/s11248-007-9134-7 | |||||
関連名称 | 10.1007/s11248-007-9134-7 | |||||
出版タイプ | ||||||
出版タイプ | AM | |||||
出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||
WoS | ||||||
表示名 | Web of Science | |||||
URL | http://gateway.isiknowledge.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=ShinshuUniv&SrcApp=ShinshuUniv&DestLinkType=FullRecord&DestApp=WOS&KeyUT=000251869000013 |