Additional Questions

Question 1

Restriction Endonucleases

Answer 1

• a.k.a. restriction enzymes

- Methalated sequences on host bacteria DNA prevent cutting of host DNA

Question 2

What is TBE Buffer?

Answer 2

• This is the buffer, fills the pores in the arose gel, so that DNA fragments can diffuse through
• You add this before you place your samples into the wells

Question 3

What is Loading dye? (what is it made of and what does it do?)

Answer 3

• Glycerol: to make the sample sink to the bottom of the well
• Bromophenol Blue: this enables us to see the sample, and tell when it is reaching the end of the arose gel, so we know when to stop electrophoresis

Question 4

Cutting sequence of Alu 1

Answer 4

AG CT

TC GA

Question 5

Cutting sequence of Taq 1

Answer 5

T CGA

AGC T

Question 6

Cutting sequence of EcoR1

Answer 6

G AATTC

CTTAA G

Question 7

Cutting sequence of Not 1

Answer 7

GC GGCCGC

CGCCGG CG

Question 8

Ethidium Bromide: what does it do, what colour is it?

Answer 8

• Used to visualise the DNA

- when bound between bases, it takes on a pink-orange fluoresce under UV light

Question 9

Transaminases: cofactor? what do they do?

Answer 9

• Pyridoxial phosphate: from Vitamin 6

- Move amino groups from amino acid and oxo acids

Question 10

What was the transaminase reaction we did? And with what enzyme?

Answer 10

• Glutamate (A.A.) + Pyruvate (keto.a.) –> Alanine (A.A.) + Oxoglutarate (keto.a.)
• Glutamate amino transferase

Question 11

Glutamate Dehydrogenase: cofactor? what does it do?

Answer 11

• Catalyses oxidative deamination of glutamate into oxoglutarate
• NAD+ and NADP+ as a cofactor

Question 12

What does aminopeptidase do?

Answer 12

• Cleaves the amino acid off the N terminus

Question 13

What does carboxypeptidase do?

Answer 13

• cleaves the amino acid off the C terminus

Question 14

2,4,-DNP

Answer 14

• Stains keto/oxo acids yellow

Question 15

Ninhydrin + Heat

Answer 15

• Stains Amino Acids purple

Question 16

Km

Answer 16

• Measure of affinity of an enzyme for its substrate
• [S] that half saturates the enzyme
• Half Vmax
• Smaller Km means larger affinity
• units mmol

Question 17

Vmax

Answer 17

• Max velocity an average enzyme can obtain

- units µmol/min

Question 18

Kcat

Answer 18

• Catalytic constant - turnover number

- I.e. # of substrate molecules transformed per molecule of enzyme per second

Question 19

Competitive Inhibitor

Answer 19

• Inhibitor and substrate similar structure
• Both compete for same active site
• Km increases, Vmax remains the same

Question 20

Uncompetitive Inhibitor

Answer 20

• When the substrate binds, this causes the inhibitors binding site to appear
• Can only bind to ESC
• Vmax is dramatically reduced - not much free enzyme to catalyse
• Km - minimally reduced