1. EDVOKIT#300: Blue/White Cloning of a DNA Fragment
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:
0.5-10µl and µl 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

2. Experiment Overview
Ligation
Transformation
EDVO page 7

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

4. Plate E.coli on medium containing:
-Amp: select for transformed cells
-Xgal: turns blue when hydrolyzed by gal enzyme
-IPTG: induces promoter
*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

5. Edvo pg 14

6. V1 µl = (final conc. X 50µl) ÷ stock conc.
PCR: Supplement pg 97
C1 X V1 = C2 X V2 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.

7. EDVOKIT#300: Blue/White Cloning of a DNA Fragment
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:
0.5-10µl and µl 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