Cloning And Vectors

Question 0

Three key features of cloning vector

Answer 0

Origin of replication
Antibiotic resistance
Unique restriction sites

Question 1

1OD of DNA
1OD of RNA
1OD of ssDNA

Answer 1

50
40
33

Therefore [DNA] = absorbance x dilution factor x above value.

Question 2

Bond between nucleotides

Answer 2

Phosphodiester bond

Question 3

What is cloning

Answer 3

Insert fragment of DNA into another DNA molecule that can self replicate in order to reproduce fragment of DNA

Question 4

Why clone

Answer 4

• Determine structure of DNA
• Sequence DNA
• express and study protein product
• Study promoter region

Question 5

What is a vector?

Answer 5

Self replicating DNA that is independent of chromosomal DNA.

Question 6

Introduction of DNA into bacteria.

Answer 6

TRANSFROMATION

Question 7

Types of vectors

Answer 7

Plasmid
Phages
Cosmid
Bacterial artificial chromosome
Yeast artificial chromosome

Question 8

Addition feature needed in mammalian vectors

Answer 8

Strong promoter (eg CMV promoter)

Question 9

Types of reporters

Answer 9

B-gal
Luciferase
GFP
CAT
SEAP

Question 10

How to prevent vector closing on itself

Answer 10

Phosphatase (calf intestine phosphatase) will remove 5’ phosphate.

Question 11

What is directional cloning?

Answer 11

Use of heterologous ends. Vector cut with two different enzymes. These enzymes also used to cut insert. Ensures correct orientation.