2011 基因工程技術原理實驗進度表
值日組 Check in* Culture E. coli DH5 (pCU1) in 3 ml LB (Amp), and E. coli DH5 (pBluescript SK+) in LB
(Amp) at 37oC overnight for plasmid DNA isolation.(前一天養菌,隔天收菌進行 Exp.3) (1) Plasmid (pCU1 and pBluescript SK+) isolation (Exp. 3),
(2) Restriction enzyme digestion (Exp. 5)
(隔天收並存於 4oC 供下一周使用，plasmid 存於-70oC 供下周使用)
G 1
(1) Gel electrophoresis (Exp. 6), and construction of pCU1 restriction map (2) Restriction enzyme digestion of pCU1 and pBluescript SK+ (vector) (Exp. 7) (隔天收並存於 4oC 供下一周使用)
G2
(1) Gel electrophoresis, and extraction of a DNA fragment from agarose gel. (Exp. 7).
(2) pBluescript SK+ phenol extraction, and ethanol precipitation (存於 4oC 供下一周使 用)
G3
* Culture E. coli DH5 in 5 ml LB at 37oC overnight for competent cell preparation.(實
驗前一天準備)
(1) CIAP of pBluescript SK+ and fill-in DNA fragment from pCU1 (Exp. 8).
(2) Ligation of a DNA fragment from pCU1 to pBluescript SK+ vector with CIAP treated
(Exp. 9). (存於 4oC 供下一周使用). 6
(3) Competent cell preparation (Exp. 10). (存於-70oC 供下一周使用)
G4
(1) Transformation by heat shock and plate on LA (Amp). Incubate at 37oC overnight (Exp.11). (隔天觀察並存於 4oC)
G5
(1) Amplified DNA fragment and check the results of ligation by PCR (Exp. 12) (存於-70oC 供製備 DNA Probe 使用).
G6
實驗期中考 G1
期中考週
(1) Total DNA isolation (Exp.13), restriction enzyme (EcoRI or HindIII) digestion.
(隔天收並存於 4oC 供下一周使用).
G2
(1) Gel electrophoresis, and transfer DNA from gel to membrane for Southern blot analysis (Exp. 15). (隔天收並存於 4oC 供 hybridization 使用)
(2) DNA probe preparation (Exp. 16) (隔天收並存於 4oC 下一周沉澱).
G3
(1) DNA probe preparation (酒精沉澱) (Exp. 16)
(2) Dot blotting. (Exp.14), (membrane 保存於 4oC 以供後續實驗)
G4
* Culture E. coli BL21 (DE3)(pET29a-A) in 3 ml LB (kanamycin), at 37oC overnight.
(前一天養菌,隔天收菌進行 Exp.18).
(1) Gene expression in E.coli and SDS polyacryamide gel electrophoresis of protein.
(Exp.18)(SDS-page 作好存於 4oC ,sample 煮沸後存在-70oC 供下一周電泳使用).
G5
(1) SDS polyacryamide gel electrophoresis of protein. (Exp.18).
(2) Southern hybridization (Exp.16 , Exp. 14) (隔天收並存於 4oC 下一周呈色).
G6 (1) Color development of Southern blotting and dot blotting.(Exp. 17 )
(2) check out
實驗數據及實驗分析（各組上台報告）
實驗期末考
