酵母双杂交实验2009.pptVIP

酵母双杂交技术—蛋白相互作用的发现及验证 2009-10-28 * * 酵母双杂交原理 GAL UAS Minimal promoter reporter gene DNA-BD AD Transcription DNA-BD AD 1aa 147aa 768aa 881aa GAL UAS Minimal promoter reporter gene DNA-BD Bait protein AD Library protein 酵母双杂交原理 AD GAL UAS Minimal promoter reporter gene Transcription DNA-BD Bait protein Library protein Reporter gene mRNAs Reporter constructs in yeast strain AH109 Vector Information BD AD 酵母表型验证 钓饵质粒构建 细胞毒性检验 自动激活检验 共转化酵母 His及Ade营养初筛 β-Gal显色复筛 抽提混合质粒 分离纯化AD质粒 质粒重新 转化酵母验证 阳性克隆 测序获取基因 文库构建或扩增 LiAc法转化酵母AH109： 一、制备AH109菌感受态： 1. Inoculate 1 ml of YPDA with several 2–3 mm colonies. 2. Vortex vigorously to disperse any clumps. 3. Transfer cells to a flask containing 50 ml YPDA 4. Incubate at 30°C for 16–18 hr with shaking (250 rpm) to stationary phase (OD6001.5). 5. Transfer overnight culture (enough to produce an OD600 = 0.2–0.3) into 300 ml YPDA 6. Incubate at 30°C for 3 hr with shaking (230–270 rpm). The OD600 will be 0.5 ± 0.1. 7. Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature. 8. Discard the supernatant and resuspend cell pellets by vortexing in this volume of sterile 25–50 ml H2O 9. Pool cells centrifuge at 1,000 x g for 5 min at room temperature. 10. Decant the supernatant. 11. Resuspend the cell pellet in 1.5 ml freshly prepared, sterile 1X TE/LiAc 12. Prepare PEG/LiAc solution. 10 ml 13. In the 1.5 ml tube, add and mix the following: ? DNA-BD/baita 0.1 ug （1.5ul） ? AD/library 0.1 ug （1.5ul） ? Herring testes carrier DNA 0.1 mg （10ul） 14. Add 0.1 ml yeast competent cells and mix well by vortexing. 15. Add 0.6 ml sterile PEG/LiAc solution and vortex at high speed to mix. 16. Incubate at 30°C for 30 min with shaking (200 rpm). 质粒共转化AH109菌： 17. Add 70 ul DMSO, Mix well by gentle inversion or swirling. Do not vortex. 18. Heat shock for 15 min in a 42°C water bath. 19. Chill cells on ice for 1–2 min. 20. Centrifuge cells for: 5 sec at room temperature at: 14 K rpm 21. Remove the supernatant. 22. Resuspe