Cloning and Western Blotting

Question 1

What does gene cloning enable?

Answer 1

Enables recombinant DNA to be inserted into a host and produce many copies

Question 2

What is recombinant DNA?

Answer 2

Artificially created DNA molecules that bring together sequences

Question 3

What are cloning vectors?

Answer 3

Plasmids that enable selection and contain many restriction sites

Question 4

Once a vector is purified, how to insert our gene/sequence of interest into it?

Answer 4

Must use restriction enzymes

Question 5

What are restriction enzymes?

Answer 5

Recognise specific sequences of DNA and cleave them

Question 6

What are restriction sites?

Answer 6

Specific DNA sequence that is recognised

Question 7

What can restriction enzymes leave?

Answer 7

Blunt end (straight cut) or sticky ends (w/ an overhang)

Question 8

Can blunt ends be ligated together?

Answer 8

Yes

Question 9

True or false: For sticky ends, two molecules must be complementary in sequences?

Answer 9

Yes

Question 10

How are fragments and vectors ligated?

Answer 10

Both are cut from same enzyme
Will be joined together by DNA ligase which catalysed phosphodiester linkage

Question 11

How is recombinant DNA used?

Answer 11

Inserted into a host and amplified

Question 12

What is transformation?

Answer 12

Process by which a host takes up cloning vector which enables bacteria to exchange genetic material

Question 13

What is the process of transformation?

Answer 13

Calcium chloride added to bacterial cell to make porous
Cells heat shocked to make them take DNA

Question 14

How to identify what cells are transformed?

Answer 14

Plasmids contain ABR genes
Cells are plated on antibiotic-containing media

Question 15

What is manipulation of genes?

Answer 15

Site-directed mutagenesis is a technique that enables manipulation of gene sequence

Question 16

Explain protein production

Answer 16

Cloning enables much higher yield of protein

Question 17

What does western blotting enable?

Answer 17

Enables detection proteins

Question 18

What is SDS-PAGE used for?

Answer 18

To denature proteins (SDS) and seperate by polyacrylamide gel electrophoresis (PAGE)

Question 19

How is SDS-PAGE visualised?

Answer 19

Using Coomassie blue

Question 20

Explain transfer to membrane step

Answer 20

Gel put into contact w/ membrane to move proteins

Question 21

Explain the block step

Answer 21

Use blocking buffer (milk, BSA) to ensure there are no free spaces on membrane for antibody to non-specifically bind to

Question 22

Explain anti-body based detection

Answer 22

Primary antibody to protein of interest
Secondary antibody to 1 antibody
Chemiluminescence