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 COOH   On the plasmid On the chromosome

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

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

Let’s Clone Something!!!!

X Ignore this alpha-beta-gal lac promoter

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DNA ligase

CaCl2 and Electroporation 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?

5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 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