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You will now test -galactosidase activity at subsaturating ONPG concentrations.

ID: 478946 • Letter: Y

Question

You will now test -galactosidase activity at subsaturating ONPG concentrations. Your instructor will assign one of the following ONPG concentrations for you to test. Calculate how you will make the 3 ml solution you need for each concentration. Note that it is critical for -galactosidase to function in as constant an environment as possible. As we dilute the ONPG, it is important not to dilute any of the other components of the assay buffer. So a -ONPG solution described below is used as the diluent.

ONPG Concentration

Dilution factor from 2.3 mM

Volume of + ONPG solutiona

Volume of - ONPG solutionb

2.3 mM

no dilution

3 ml

0 ml

2

5

10

a + ONPG solution = 2.3 mM ONPG, 0.1 M sodium phosphate pH 7, 2 mM MgSO4, 0.1 M -ME

b - ONPG solution = 0.1 M sodium phosphate pH 7, 2 mM MgSO4, 0.1 M -ME

ONPG Concentration

Dilution factor from 2.3 mM

Volume of + ONPG solutiona

Volume of - ONPG solutionb

2.3 mM

no dilution

3 ml

0 ml

2

5

10

Explanation / Answer

ONPG Concentration Dilution factor from 2.3mM Volume of + ONPG solution a Volume of - ONPG solution b 2.3 mM No dilution 3 mL 0 mL 1.15 mM 2 3 mL 3 mL 0.46 mM 5 3 mL 7.5 mL 0.23 mM 10 3 mL 15 mL

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