L3 Handout - Lab 3 - Transformation & Enzymes

L3 Handout - Lab 3 - Transformation & Enzymes - Bio...

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Bio 1A Lab Lecture Outline # 3 Transformation & Enzymes Sept. 15, 2008 I. Cloning & Ligation note: steps will become clearer each week. A. DNA structure B. Overview- Isolate DNA of interest and vector DNA C. Restriction Digest (Cut) D. Ligate, Transform , Screen (various ways) and analyze! For example – Insulin gene Host cells with vector Human tissue fragment cells to release DNA fragment cells to release DNA follow steps to isolate vector DNA follow steps to isolate DNA Isolated vector DNA Isolated source DNA II. Transformation- uptake and expression of DNA in a host cell A. Host cell = Escherichia coli ( E. coli ) a. Growth & Division (LB =energy source, carbon source, elements). b. LB plates – LB with agar, one bacterium to a colony. c. selectable genes in the vector – eg. ampicillin affects cell wall synthesis. B. Transformation = rare event. How do you screen for this? for various classes of DNA? a. Uptake and expression of vector genes and insert, screening & pBLU. b. Transformation protocol III. Light Spectroscopy A. Overview B. Identification – Absorption Spectrum C. Quantification – (A = clZ) D. Blank – why?
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