Cloning

Question 1

Standard cloning

Answer 1

Cloning is directional as we know what way round to insert the fragment into the vector, since there are only two enzymes present

Restriction sites remain intact as the same enzyme can cut the site again

High ligation efficiency as fragment can’t self-ligate since enzymes on each end aren’t the same or complimentary

Low vector self ligation as enzymes on each end of the vector aren’t the same or complimentary

Question 2

Complimentary cloning

Answer 2

Cloning is directional as fragment will only insert one way round

Restriction sites may not remain intact as the two enzymes used to join one point aren’t the same

High ligation efficiency because enzymes on each end of the fragment aren’t the same or complimentary

Low vector self ligation as enzymes in each end of the fragment aren’t the same or complimentary

Question 3

Blunt ended cloning

Answer 3

Cloning is non-directional as the fragment can fit in any way round as it isn’t sequence dependant

Restriction sites lost as complimentary nucleotides fill in 5’ and 3’ overhangs

High vector self ligation as enzymes on each end of the vector are the same

Advantages include: flexible and versatile
Disadvantages: low efficiency/yield