Tube Amount Transferred 0.5ml (500ul) 0.5ml (500ul) 0.5ml Buffer Amount 4.5 ml S
ID: 202262 • Letter: T
Question
Tube Amount Transferred 0.5ml (500ul) 0.5ml (500ul) 0.5ml Buffer Amount 4.5 ml Step Dilution 1/10 Final Concentration 1/10 Source Stock Tube 1 Tube 2 Tube 3 4.5 ml 1/10 4.5 ml 1/10 1/100 1/1000 1/10000 4.5 ml 1/10 (500ul) 4 0.5ml (500ul) Do not discard any of the tubes 1-4 as they will be used for Experiment 2. 5. Obtain 5 cuvettes. For one cuvette, add 1 ml of 2.5mM ONPG solution and 1 ml of stock enzyme solution. Carefully watch the cuvette until it turns a bright yellow (not pale yellow). St the reaction by adding 2 ml sodium carbonate. Use this cuvette to determine the a maximum of the solution containing ortho-nitrophenolate. op bsorption (3) Why is this important? Use this wavelength for the remainder of your experiments. (4) Wavelength = 6. Blank the spectrophotometer at the determined wavelength. (5) What would you use as your blank? 7. For the remaining cuvettes, add 1.5ml of 2.5mM ONPG solution to each. In one cuvette, add 1.5ml of enzyme solution from tube 1 (1/10 dilution) to the ONPG and measure absorbance every 15 seconds for 5 minutes. Repeat for the other dilutions (Tubes 2-4; 1/100-1/10000). Record data in Table 2Explanation / Answer
3) The potential to quantitate action of b-galactosidase is of great need in molecular biology. ILacZ gene used as a reporter for the expression of gene , because is not found in mammalian cells generally. ONPG hydrolysis i.e, catalyzed by an enzyme beta galactosidase leads to the formation of coloured product absorb maximum at 400nm approx. The efficacy of absorbance at 420 nm, that is around the maxima, permits an exact evaluation of hydrolyzed product without any absorbance by non-hydrolyzed substrate at the background.
Blank is used to differentiate it from a reaction buffer and set blank as a reference for reading the absorbance of all different sample.
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