Download + IPTG + X-gal

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
Blue/White Cloning Vectors
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-galactosidase
H2N

On the plasmid

On the chromosome
COOH
-Complementation
 -gal
MCS
 -gal
Nonfunctional
chromosome
Nonfunctional
E. coli engineered
for Blue/White Screening
-Complementation
 -gal
 -gal
MCS
Functional
chromosome
E. coli engineered
for Blue/White Screening
Let’s Clone Something!!!!
X Ignore this
lac promoter
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA ligase
Transformation
CaCl2 and Electroporation
Transformation using CaCl2-Competent Cells
+ IPTG
+ X-gal
90 seconds
Transformation using Electroporation
+ IPTG
+ X-gal
LB broth
Electroporator
What is Needed for the Blue/White Screen?
X-gal
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
Ampicillin
-lactam ring
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.)
PCR
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
URL for PCR Animation:
www.dnalc.org/shockwave/pcranwhole.html