Lecture 9/11 - E2

Question 1

What is a genomic DNA library?

Answer 1

All the nuclear DNA represented in many individual DNA clones

Question 2

What are multiple cloning sites (MCS)?

Answer 2

Many different restriction sites arranged next to one another
- Where the piece of DNA has been inserted

Question 3

What is the role of B-lactamase?

Answer 3

It is an enzyme that destroys penicillin-like compounds and confers resistance to ampicillin

Question 4

What is the size of 1 DNA ApaI (10,090) - Xhol (33498) restriction site ends?

Answer 4

Take the distance between the two:

33,498-10,090 = 23,408 base pairs

Question 5

What is bacterial trsnformation of recombinant DNA?

Answer 5

Where each transformant colont is clonal and has only one type of piece of cloned DNA in the plasmid (but lots of copis of that particular plasmid)

Question 6

Insertional Inactivation

Answer 6

Where you place your foreign DNA into an existing gene on the plasmid and destroy that genes function
- Allows detection of successful cloning events by visual inspection of colonies

Question 7

Example of insertional inativation with the lac Z gene

Answer 7

The MCS is within the lac Z coding sequence. Insertion of foreign DNA into the MCS inactivates lac Z and blocks characteritic expression

Question 8

Importance of the Lac Z gene

Answer 8

Encodes proteins produced by the lac operon

Expression produces the B-galactosidase protein required for E. coli to use lactose and grow

Question 9

Importance of B-galactosidase

Answer 9

Converts lactose to glucose+galactose

Question 10

Blue vs White Colonies

Answer 10

Blue = activated lac Z gene, B-galactosidase is produced
White = Inactivated lac Z gene, B-galactosidase is not produced

Question 11

Which colonies do you want as an investigator?

Answer 11

The white colonies

Question 12

What are complementary DNA (cDNA) libraries made of?

Answer 12

Reverse transcriptase uses RNA-dependent DNA polymerase to create this library from mRNA

Question 13

What is the process of making cDNA libraries?

Answer 13

Reverse transcriptase uses a doubel stranded region to initiate synthesis
Therfore, mRNA must be primed using an oligo dT
Oligo dT is annealed to create the poly A tail

Question 14

Characteristics of a genomic DNA library

Answer 14

Contains all nuclear DNA sequences including gene coding DNA and both the transcribed/non-transcribed regulartory features
Increased frequency = larger size

Question 15

Characteristics of a cDNA libray

Answer 15

Contains only portions of the genome that are transcribed (usually just the mRNA)
Introns and non-transcribed regulatory sequences (promoters, enhancers) are not included
Increased frequency = increased mRNA

Question 16

What is PCR?

Answer 16

Polymerase Chain Reaction

Repeated DNA synthesis of a defined sequence in a test tube

Question 17

Requirements for a PCR experiment?

Answer 17

Template DNA - ds DNA
Primers - ss DNA oligos
Thermo-resistant DNA Polymerase
dNTPs
Thermocycler - incubator that can rapidly change temperature

Question 18

Steps of PCR

Answer 18

Denaturation - temp increase to seperate DNA strands
Annealing - temp decrease to allow primers to base pair to the template
Extension - Polymersase extends the primer to form DNA strand