Lab 21 Goals and Objectives:

Exercise 59: Bacteriological Examination of WaterConfirmed Test: check EMB plate for coliforms

EDVOKIT#300: Blue/White Cloning of a DNA FragmentTransform E. coli with your ligation reactions (pg 12-13)Each group will need:

0.5-10µl and 100-1000µl micropipettorsTips: large and small2-1.5ml tubes containing pellets of E. coli on iceCaCl2 on iceRB (recovery broth)2 tubes of glass beads2 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