Lab 21 Goals and Objectives:

EDVOKIT#300: Blue/White Cloning of a DNA Fragment Transform E. coli with your ligation reactions (pg 12-13)

Each group will need:

0.5-10!l and 100-1000!l micropipettors

Tips: large and small

2-1.5ml tubes containing pellets of E. coli on ice CaCl2 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 97

Ligation

Transformation

EDVO page 7

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

Amy Warenda Czura, Ph.D. 1 SCCC BIO244 Lab 21 Notes

Edvo pg 14

C1

Concentration

of stock

solution or

reagent:

X stock or

mM/!M stock indicated

V1

How much

of the stock

reagent you

need: this is what you

are solving

for! in !l

C2

Concentration

of the reagent

in the final

solution:

1X or mM/!M

concentration indicated

V2

Volume of the

final solution:

in !l

PCR

reactions are

50!l

C1 X V1 = C2 X V2

Solve for V1

V1 = (C2 X V2) ÷ C1

V1 !l = (final conc. X 50!l) ÷ stock conc.

C1 X V1 = C2 X V2

PCR: Supplement pg 97

Lab 21 Goals and Objectives:

EDVOKIT#300: Blue/White Cloning of a DNA Fragment Transform E. coli with your ligation reactions (pg 12-13)

Each group will need:

0.5-10!l and 100-1000!l micropipettors

Tips: large and small

2-1.5ml tubes containing pellets of E. coli on ice CaCl2 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 97

Amy Warenda Czura, Ph.D. 2 SCCC BIO244 Lab 21 Notes