M5 L15

Question 1

gene cloning aims

Answer 1

obtain isolated and purified copies of specific gene sequences

Question 2

cloning genes allows

Answer 2

• sequence and structure determination
• gene function leads to
• gene manipulation

Question 3

clone

Answer 3

a large number of identical cells ot molecules with a single ancestral cell or molecule

Question 4

cloning vectors

Answer 4

a carrier DNA molecule that allows attached DNA to be replicated in a cell

Question 5

restriction enzyme

Answer 5

an enzyme that recognises and cleaves DNA at specific sequences

Question 6

DNA ligase

Answer 6

an enzyme that can covalently link DNA molecules by creating phosphodiester bonds

Question 7

cloning decisions

Answer 7

insert?
vector?
what next?

Question 8

plasmids as cloning vectors

Answer 8

ideal because
- well characterised
(sequence and function of genes known)
- small and easy to purify and manipulate

Question 9

plasmid vector useful features

Answer 9

-ori
-selectable marker
-unique restriction enzyme cleavage sites
- easy screening methods
- often have high copy number

Question 10

cloning steps

Answer 10

restriction
ligation
transformation
selection

Question 11

CaCl2 transformation induction

Answer 11

cells are made competent by treatment with ice cold CaCl2 then heat-shocked at 42 deg.

Question 12

electroporation

Answer 12

cells are made competent with an electric field (often 2500V)