PRE-LAB 4 Enzyme Kinetics Pre-laboratory Assignment Directions: TYPE the answers

Question

PRE-LAB 4 Enzyme Kinetics Pre-laboratory Assignment Directions: TYPE the answers to 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. Questions: For questions #1 through #4, please put your answer on the table below. 1. (1pt) 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 half pill. How many mL of phosphate buffer do you need to add to a half pill? 2. (Ipt) 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. 3. (Ipt) After your 1:2 dilution of ONPG, what is the concentration of ONPG? 4. (1pt) For each tube, how long will the enzyme be allowed to react with substrate? Answer Question 1. volume phosphate buffer? 2. mL of ONPG 3. concentration of ONPG? 4. total length of reaction? mL mL water mM Seconds mL ONPG+

Explanation / Answer

1. 50 units per 1ml

Half a pill will have about 4500 units (9000/2),

Buffer needed to dissolve 4500 units = 1/50*4500 =4500/50 = 90 ml

To cross check, for 1 pill i.e. 9000 units you would require 9000/50 = 180 ml

Hence, we have calculated half the amount of buffer needed for half a pill

2. 1:2 dilution of ONPG where 1 is ONPG 20mM and 2 is water, then

2ml of ONPG and 4 ml of water

3. C1V1=C2V2

20mM*2ml = x(6ml)

6.67mM

4.

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