Lecture 20

Question 1

How do you clone PCR products?

Answer 1

-Restriction cut sites can be created on PCR product ends by adding to the ends of the PCR primers

• Taq DNA pol adds on an ADENINE at the 3 prime end of the PCR product (adenine overhang)
• The vector has a 3 prime THYAMINE overhang

-The vector must have a matching overhang

Question 2

Can the vector religate itself?

Answer 2

No, so only clones with inserted DNA can survive

Question 3

What is Gibson Assembly?

Answer 3

A process that can rapidly assemble multiple (more than 10) DNA fragments in one reaction

Question 4

Describe the process of Gibson Assembly

Answer 4

1. 5 prime Exonuclease ‘chews’ back the 5 prime end, creating complementary fragments
2. DNA fragments anneal
3. DNA pol extends at the 3 prime end
4. DNA ligase seals nicks

Question 5

For Gibson Assembly to work what must the DNA have?

Answer 5

Overlapping sequences at one end

Question 6

If a DNA sequence is hard to PCR/clone or it doesn’t exist in nature what can we do?

Answer 6

1. Buy the synthesised DNA in a vector

2. Buy synthesised lengths of DNA g-blocks (up to 3kb)