Past exam questions

Question 1

Restriction endonuclease

Answer 1

Digest (cut) DNA within a specific nucleotide sequence (recognition site)

Question 2

Molecular cloning: 1. Amp gene 2. lacZ gene 3. MCS 4. Restriction endonuclease sites

Answer 2

1. Gene for selection: Antibiotic resistance 2. Reporter gene (IPTG will induce expression of beta­galactosidase from lacZ gene) 3. region where foreign DNA can be inserted 4. unique RE sites for restriction mapping

Question 3

Method of transformation:

Heat shock method

Answer 3

Question 4

Method of transformation:

Electroporation method

Answer 4

Question 5

Antibiotics in propagation?

Answer 5

Inoculate growth medium (such as Luria broth) containing antibiotic (eg. ampicillin) with positive clone (cells containing the recombinant DNA)

Antibiotic used in the growth medium depends on the type of antibiotic resistance gene within the recombinant DNA plasmid

Question 6

Induction of protein

Answer 6

Using the lac repressor in vitro

● can exploit this system for controlled regulation of protein

expression in vitro

● Need to provide:

○ lacI gene plus promoter and operator sequence (expression vector)

● IPTG: bind to lac repressor protein and release from operator, therefore ability to control transcription to regulate lac repressor protein

Question 7

Describe Western Blot analysis (inc. reagents)

Answer 7

1. Coat surface with sample (antigens)
2. Block unoccupied sites with non­specific protein
3. Incubate with primary antibody against specific antigen
4. Incubate with secondary antibody­enzyme complex that binds primary antibody
5. Add substrate
6. Formation of coloured product indicates presence of specific antigen

Question 8

SDS-PAGE denaturation buffer. Explain the purpose of each reagent.

1. Bromophenol blue dye
2. glycerol
3. SDS (sodium dodecyl sulphate)
4. DTT (dithiothreitol)

Answer 8

1. enables monitoring
2. provides density
3. SDS (sodium dodecyl sulphate)

• on avg. one SDS binds fro every 2 AAs
• Binds proportional to Mr of protein
• contributes large net negative charge

4. A reducing agent (eg. DTT or beta­Me)
   * reduces disulphide bonds