CB - DNA Technology

Question 1

How can DNA fragments be separated and visualised?

Answer 1

Separated by Gel Electrophoresis
Visualised with fluorescent dyes and UV light

Question 2

How are DNA fragments joined?

Answer 2

DNA Ligase + ATP

Question 3

What are 5 ways to construct a recombinant DNA molecules?

Answer 3

1. Cut vector and gene with same restriction enzyme
2. Cut vector and gene with 2 different restriction enzymes
3. No compatible enzyme available
   -Use nucleases to create blunt ends on gene and vector
   -Use DNA Polymerase to fill in ends on gene and vector
4. Use of linkers/adapters
5. TA cloning

Question 4

What are the 3 steps of introducing DNA into living cells?

Answer 4

1. Preparing competent bacteria cells
2. Introducing DNA into cells
3. Select only the cells that have taken up the recombinant DNA

Question 5

How is a competent E.Coli cell prepared?

Answer 5

1. Normal bacterium is treated with 50mM CaCl2 and the plasmid binds to the exterior
2. The competent cell is heat shocked and the plasmid is transported into the cell

Question 6

What are 3 methods of distinguishing between bacteria that have taken up recombinant DNA or self ligated vector?

Answer 6

1. Replica Plating
2. Blue/white selection
3. Restriction mapping

Question 7

Why would a bacterial cell be chosen over an animal cell?

Answer 7

1. Divides much quicker
2. Can grow in liquid media

Question 8

Why would a animal cell be chosen over an bacterial cell?

Answer 8

Able to glycosylate proteins