Gene Cloning

Question 1

Give 2 reasons for cloning a gene

Answer 1

To be able to mutate it
To over-express it

Question 2

What process is used to lyse the cell in the first step of DNA extraction

Answer 2

Mechanical or chemical lysis

Question 3

In the second step of DNA extraction the hydrophobic and hydrophilic components (which one is DNA) need to be separated - what is added in a 1:1 ratio

Answer 3

DNA is hydrophilic
Phenol and chloroform

Question 4

DNA is precipitated then centrifuged in the third step of DNA extraction - how is it precipitated

Answer 4

Adding cold alcohol

Question 5

What is the last step in DNA extraction

Answer 5

Purification

Question 6

There are 3 temperatures used during PCR - what are they - DAE

Answer 6

Denaturation of 95 - Breaks helix into 2 separate strands
Annealing of 50 - Allows synthetically synthesised primers to H bond to their complementary regions on the template strand
Extension temperature of 68-72 - Allows Taq polymerase to bind primers and synthesise DNA in a 5’ 3’ direction

Question 7

What are the 5 requirements to carry out PCR

Answer 7

• DNA template
• DNA primers
• Taq polymerase
• dNTPs - free deoxyribonucleoside triphophates
• Buffer

Question 8

What is the purpose of a cloning vector

Answer 8

Stabilise the PCR amplified DNA sequence inside a host

Question 9

What are the 3 main features of plasmid vectors

Answer 9

Origin of replication
Antibiotic resistance gene - selectable marker
Multiple cloning site - facilitate insertion of PCR product

Question 10

During ‘cut and paste’ which enzyme causes a break at a specific sequences - what are these sequences called

Answer 10

Restriction endonuclease - restriction sites

Question 11

What are the 2 different overhangs that can be produced

Answer 11

Sticky ends and Blunt ends

Question 12

Which enzyme restores the backbone in the plasmid

Answer 12

DNA ligase

Question 13

What is the process of inserting recombinant DNA into host cells called

Answer 13

Transformation

Question 14

What process do cells have to go through in order to make them take in extracellular DNA

Answer 14

They need to be made competent

Question 15

What are the 2 methods of transformation

Answer 15

Chemical transformation
Electroporation

Question 16

In chemical transformation what are the cells chilled in the first step, what increases permeability in the second step and what facilitates the uptake of the plasmid through the PM in the third step

Answer 16

Cations
Heat shock
Cations

Question 17

In electroporation what increases the membrane permeability in the first step and how are the cells made electro-competent in the second step

Answer 17

High-voltage electric shock
Chilling and washing with glycerol to remove any ions/chemicals

Question 18

What do selectable markers provide

Answer 18

Mechanism for discriminating between transformed and non-transformed host cells

Question 19

Give 2 reasons for recovery after transformation

Answer 19

Restoration of membranes after shock treatment
Opportunity to express antibiotic resistant gene