Cloning 2

Question 1

Describe the 4 properties of DNA vectors

Answer 1

1. Replicate autonomously in bacteria
2. Contain unique restrction endonucelase cleavage sites that are only present once
3. Carry a selectable marker (usually antibiotic resistance genes) to distnguish host cells that carry the vector and those that do not
4. Relatively easy to purify from bacterial chromosome DNA

Question 2

What two things do you consider when chossing a vector?

Answer 2

depends on the insert size and application

Question 3

Types of vectors 1-4 increasing size

Answer 3

Plasmid - <10kb
Phage 5-20kb
Cosmid 35-45 kb
BAC (bacterial artificial chromosome) 75-300kb

Question 4

Define plasmid vectors

Answer 4

naturally occuring extra-chromsomal double stranded curcular DNA that has an Origin of replication
It has been modified to conain an antbiotic resitance gene and a multiple cloning site

Question 5

4 steps of construction of a recombinant DNA molecule

Answer 5

1. Plasmid vector is cut with restircution enyme at the MCS to produce cohesive ends
2. DNA insert is prepared such that it is cut with the same enxyme to produce the same cohesive ends
3. annealing of DNA insert to plasmid vector by pase pairing at the compatible cohesive ends
4. Ligase gorms covalent bonds to join FNA bacbones of DNA insert and vector

Question 6

Why is ligation not 100% efficient?

Answer 6

The two ends of the vector can readily ligate with eachother

Question 7

What are the two ways that we can solve the ligation of the two ends of the vector?

Answer 7

1. Reduce self ligation by treating with a phosphotase that removes the phosphodiester bond
2. Cut the vector with 2 different restriction enzymes to create non-complimentary sticky ends

Question 8

What are the 2 advantages of using two different restriction enxymes?

Answer 8

1. Prevents self ligation by creating two non-complimentary sticky ends
2. Allows the insert be be inserted with a known orientation