Cloning

Question 1

What are the steps in sub-cloning

Answer 1

Cloning-
Isolating DNA sequence of interest (insert) with restriction enzymes

Insert is purified using RNase or specific DNases (gel isolation)

Number of copies is amplified through PCR

Insert is ligated into vector which has been digested by restriction enzyme (3:1 ratio to prevent re-ligation)

Insert and vector transformed into bacterial cell

Then bacteria cells are screened to see which ones have taken up desired DNA

Question 2

How does blue white screening work

Answer 2

Bacteria contains a deletion mutant of lacZ which is restored to function by partial lacZ gene in plasmid

If the insert has binded to vector then you won’t have the partial lacZ gene in plasmid

Once you plate bacteria with X-gal it will turn blue if you didn’t get transformation and stay white if you did

Question 3

How single restriction used in cloning

Answer 3

Use a single restriction enzyme to cut both the vector and insert
Used when the orientation of the insert is unimportant

Question 4

How is double digests restriction used in cloning

Answer 4

Uses two different restriction enzymes to cut insert and vector
Used to allows the insert to be cloned directionally as vector and insert can only join in one direction

Question 5

How does compatible end restriction work

Answer 5

Uses restriction enzymes that generate compatible ends on vector and inserts so they can be ligated in specific orientation

Question 6

What are the pros and cons of blunt end cloning

Answer 6

Pros-
Very versatile (all termini by blunt end cutters will be compatible for ligation)

Cons-
Not very efficient as it requires high concentration of substrates
Inserts can be inserted in any orientation
High probability of re-ligation

Question 7

How does restriction based cloning work

Answer 7

Uses restriction enzymes to cut DNA at specific sites allowing it to be inserted into vector
Used for sub-cloning, directional cloning

Question 8

How does PCR based cloning work

Answer 8

Uses PCR amplification to generate DNA fragments for subsequent cloning without the need for restriction digestion or phosphorylation

Used for rapid and efficient DNA fragments for cloning

Question 9

How does recombination based cloning

Answer 9

Rely on site specific recombination systems to facilitate directional transfer of DNA fragments between vectors (Gateway cloning)

Used for efficient and seamless insert transfer without the use of restriction enzymes (high throughput)

Question 10

What is TA cloning

Answer 10

Taq- polymerase adds a single 3’- adenine overhang to PCR product which is complementary to the 3’- thymidine.
Vector is treated with topoisonerase 1 which covalently binds to it (creates a single strand break in DNA) allowing for the insertion of the insert

Very simple fast and efficient without the need for restriction enzymes

You can get non specific products due to non specific nature of A overhang ligation

Question 11

What is golden gate assembly

Answer 11

Unique 4 base overhangs are designed for each DNA fragment to be assembled.
Fragments with complementary overhangs are annealed and ligated with T4 DNA ligase
Final product no longer contains the type 11S restriction enzyme recognition site so reaction is irreversible

Very efficient and versatile (many combinations)
But can get insert mutations as amplified through PCR