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Cloning 原理

Cloning 原理. 許嘉如. Clone and Cloning. Clone ( n ): –organism (or cell or molecule) that is genetically identical to another organism (or cell or molecule) from which it is derived. –a group of replicas of all or part of a macromolecule (as DNA or an antibody).

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Cloning 原理

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  1. Cloning 原理 許嘉如

  2. Clone and Cloning • Clone (n): –organism (or cell or molecule) that is genetically identical to another organism (or cell or molecule) from which it is derived. –a group of replicas of all or part of a macromolecule (as DNA or an antibody). –the aggregate of the asexually produced progeny of an individual. • Clone (v): Genetic engineering and cloning

  3. Cloning目的 可以大量保存所建構的DNA片段

  4. 4 kinds of cloning vectors: • Plasmids • Bacteriophage • Bacterial Artificial Chromosome (BAC) • Yeast Artificial Chromosome (YAC)

  5. Two types of DNA cleavage ends

  6. Vector 的製作- TA vector Engineer the Ahd I site in two primers Primer Fwd: EcoRI-AhdI---->Primer Rev: BamHI-AdhI---> Do PCR and get this band:EcoRI-AhdI------2kb Junk-----AhdI-BamHI Insert via digestion with Eco and Bam into your plasmid. to get:Plasmid---------EcoRI--AhdI----2kb junk----AhdI--BamHI---Plasmid When you do a digest with AhdI you getPlasmid--EcoRI--GACNNT NNNGTC--BamHI--Plasmid Plasmid--EcoRI--CTGNNN TNNCAG--BamHI--Plasmid

  7. TA Vector

  8. 乳糖操縱子

  9. 結構基因─Z基因 • z基因的產物是β-galactosidase ，他催化乳糖分解成葡萄糖及半乳糖。

  10. 結構基因─Y基因 • Y基因的產物是滲透酶，此酶存在細胞膜上的膜蛋白，它的功能是將培養基中的乳糖送入大腸菌內。

  11. 結構基因─A基因 • A基因的產物是乙醯化酶，它的功能是將細胞內一些半乳糖苷加上乙醯基，避免被半乳糖苷酶(β-galactosidase)分解變成有毒物，防止細胞中毒。

  12. 乳糖操縱組的表現與乳糖有關 • 乳糖不存在時：抑制蛋白(repressor)會與操縱子(operon)結合，抑制基因表現。

  13. 乳糖存在時

  14. 藍白挑原理( Blue-White Selection ) • IPTG ( Isopropyl-β-D-thiogalactose ) : 乳糖相似物，但不會被β-galactosidase分解，為inducer • X-gal ( 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside ) : 經β-galactosidase 分解產生藍色

  15. Do PCR and get A-tail fragments TA Vector Steps of DNA cloning Two Clones

  16. Blue/White colony screening • Blue : no insert • White : have insert 0.1mM IPTG, 40μl/ml X-Gal & 50 μg/ml Amp onto LB medium

  17. Essential elements for Cloning Vector • Self-replication • Selectable markers • Unique restriction sites • Small size, easy transformed

  18. Bacteriophage as cloning vector:

  19. BAC as cloning vector

  20. YAC

  21. Application • cDNA Library 的建構 , cDNA Microarray • Subclone into expression vector • 放大及表現目標基因 • 保存目標基因

  22. Troubleshooting - No colonies • Ligation steps • Transformation steps

  23. Ligation steps • Insert size ~ 1.5 kb • Insert & vector molar ratio ~ 1 : 1 (The final DNA concentration should be ~ 10 ng/ul ) • Ensure ligation buffer or TA vector can work ~ check the background control • Ligation incubation is not long enough

  24. Transformation steps • Check the background control ~ pUC18 • Check heat shock step ~ 42 ℃, 90 sec , 37℃ incubate for 1 ~ 1.5 hrs • Competent cells lose efficiency

  25. Thanks for your attentions !!

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