1. Molecular Genetics Bio350/516
Lecture 5, 1/31/06
Today
I. Blue/White Cloning Vectors (continued)
A. Transformation
1. CaCl2
2. Electroporation
II. PCR (Polymerase Chain Reaction)
A. Enormous amplification of specific DNA sequences
III. Probe Design
A. Do solved problem in Chapter 4

2. Blue/White Cloning Vectors

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

4. -galactosidase
H2N
COOH  
On the plasmid
On the chromosome

5. for Blue/White Screening
-Complementation
 -gal
MCS
 -gal
Nonfunctional
chromosome
Nonfunctional
E. coli engineered
for Blue/White Screening

6. for Blue/White Screening
-Complementation
 -gal
MCS
 -gal
Functional
chromosome
E. coli engineered
for Blue/White Screening

7. Let’s Clone Something!!!!

9. X
Ignore this
alpha-beta-gal
lac promoter

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

11. DNA ligase

12. CaCl2 and Electroporation
Transformation
CaCl2 and Electroporation

13. Transformation using CaCl2-Competent Cells
+ IPTG
+ X-gal
90 seconds

14. Transformation using Electroporation
+ IPTG
+ X-gal
LB broth
Electroporator

15. What is Needed for the Blue/White Screen?

17. 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
X-gal
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

18. Ampicillin
-lactam ring

22. Summary of Blue/White Cloning and -Complementation
1. Cut out gene of interest with restriction enzyme
2. Cut B/W cloning vector with same restriction enzyme (MCS)
a. Dephosphorylate vector to prevent self-ligation
3. Mix insert with vector and add ligase
4. Transform E. coli that is made for B/W screening
5. Plate onto media that contains:
a. ampicillin (E. coli cells that are not transformed will not grow as they are
ampicillin sensitive – ampicillin differentiates between Ampr and
Amps transformants. This is a selection.)
b. IPTG (binds to the lac repressor and induces expression from the lac promoter)
c. X-gal (functional -galactosidase cleaves X-gal to form blue precipitate;
distinguishes between plasmids that have an insert [white transformants]
and plasmids that do not have an insert [blue transformants]. This is a
screen.)

23. PCR

24. What do we Need for PCR?
1. DNA polymerase that is resistant to heat denaturation (get one from a
hyperthermophile)
2. dNTPs
3. PCR buffer (contains Mg++; [Mg++] critical)
4. Thermocycler (Peltier device)
5. Target DNA
6. Oligodeoxynucleotide primers

25. URL for PCR Animation: