20. Restriction endonuclease 3

Question 1

how can blunt ends be made with the klenow enzyme?

Answer 1

the klenow enzyme has 3’ exonuclease and polymerase activity, therefore can join sticky and blunt ends.

Question 2

what will the klenow enzyme do if the 3’ end is the gap?

Answer 2

the klenow enzyme will fill in the gap

Question 3

what will the klenow enzyme do if the 5’ end has the gap?

Answer 3

the klenow enzyme will trim off the opposite strand 3’ end to make it shorter

Question 4

why does vector self ligation occur and how do you prevent it?

Answer 4

its required if the lineralised vector has compatible ends. DNA ligase requires a free 5’ phosphate and 3’ OH.

can use phosphatase to remove the posphate at the 5’ end to allow binding

Question 5

what is a MCS? (multiple cloning site)

Answer 5

a multiple cloning site is a short plasmid region with many restriction sites

Question 6

what is important about MCS’s? give two advantages

Answer 6

vector and insert DNA are cut with two different enzymes and there are incompatible sticky ends used for each fragment

this eliminates vector self ligation and the insert is ligated in a specific orientation