The following questions and turn in BEFORE lab begins. If pre-lab is not complet
ID: 938795 • Letter: T
Question
The following questions and turn in BEFORE lab begins. If pre-lab is not complete at the beginning of lab. the student will be unable to participate in the lab session.For questions #1 through #4, please put your answer on the table below. In this lab. we will make lactase solution that is 50 units of lactase per mL phosphate buffer. The Lactaid pill's box label states that each pill contains 9.000 units. We will be using a half pill. How many mL of phosphate buffer do you need to add to a half pill? In this lab. we will be performing a 1:2 dilution of 20mM ONPG. You will need 6ml.-You will add mL of 20mM ONPG and mL of water to your test tube. After your 1:2 dilution of ONPG, what is the concentration of ONPG? For each tube, how long will the enzyme be allowed to react with substrate? What is the final ONPG concentration (in mM) of each of the Reaction tubes ? If the normal substrate for lactase is lactose sugar, why do we use ONPG? In this assay, will we be varying enzyme concentration or substrate concentration? Why?Explanation / Answer
Solution.
1) The half of Lactaid pill contains 9000/2 = 4500 units, the concentration is 50 units/mL, so the volume is 4500/50 = 90 mL;
2) A 1:2 dilution of ONPG can be made by adding of 1 part of ONPG to 2 parts of water. The total number of parts is 1+2 = 3, so you will add 2 mL of ONPG and 4 mL of water to your test tube;
3) After the dilution, the total volume increases 3 times, so the concentration decreases 3 times becoming 20/3 = 6.67 mM;
6) Lactase splits ONPG into ONP and galactose. ONP is a clear yellow substance, so you can use a spectrophotometer to measure how much product is being produced. Meanwhile, lactase can break the lactose into glucose and galactose, which are transparent for a spectrophotometer and cannot be detected this way.
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