Lab 21 Goals and Objectives: Exercise 59: Bacteriological Examination of Water Confirmed Test: check EMB plate for coliforms EDVOKIT#300: Blue/White Cloning of a DNA Fragment Transform E. coli with your ligation reactions (pg 12-13) Each group will need: µl and µl micropipettors Tips: large and small 2-1.5ml tubes containing pellets of E. coli on ice CaCl 2 on ice RB (recovery broth) 2 tubes of glass beads 2 plates (nutrient agar with Amp, X-gal and IPTG) HOMEWORK: calculate the recipe for PCR reactions to be set up in the next lab. See the supplemental handout in the packet page 89!

Ligation Transformation EDVO page 6 Experiment Overview

Vector + gene we want to clone + ligase ~incubate~ Two possible products: -gene ligated into vector -vector religated without gene  Transform into E.coli

*gene ligated into vector -disrupts LacZ gene, -no  gal enzyme, -colonies white *vector religated without gene  -has intact LacZ gene, -produces  gal enzyme, -Xgal gets hydrolyzed, -colonies turn blue Plate E.coli on medium containing: -Amp: select for transformed cells -Xgal: turns blue when hydrolyzed by  gal enzyme -IPTG: induces promoter

Edvo pg 12-13

C1 Concentration of stock solution or reagent: X stock or mM/µM stock indicated V1 How much of the stock reagent you need: this is what you are solving for! in µl C2 Concentration of the reagent in the final solution: 1X or mM/µM concentration indicated V2 Volume of the final solution: in µl PCR reactions are 50µl C1 X V1 = C2 X V2 Solve for V1 V1 = (C2 X V2) ÷ C1 V1 µl = (final conc. X 50µl) ÷ stock conc. C1 X V1 = C2 X V2