Ethanol Lab Preparation

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Arizona State University *

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367

Subject

Chemistry

Date

Dec 6, 2023

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pdf

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3

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Lab 8 Preparation Fill in the sample table for the standard curve samples. The unknown ethanol samples and controls are also given in this document. We will also go over the unknown alcohol samples and the control samples (other than no ethanol) in class before starting the experiment. You are bring your only samples of this lab. Please follow the rules for samples provided in the lab. Write down AND taking a picture of the alcohol content (in %) of the container. Notes on Standard curve samples: Calculation all sample to have a final volume (V 2 ) of 250 l Well concentration of buffer is consistent thus volume added is consistent o 0.15 M or 150 mM stock concentration (of sodium pyrophosphate in buffer stock, there are more components but it is the sodium pyrophosphate that we are paying attention to) o Desired sample concentration is 75 mM Well concentration of NAD+ is consistent thus volume added is consistent o Stock concentration: 3 mM o Desired well concentration: 1 mM Amount of enzyme is consistent thus volume added is consistent o Stock concentration: 5000 units/ml o Desired concentration: 25-50 units/ml Choose an enzyme concentration in this range The well concentration of ethanol is DIFFERENT thus the volume of each sample will be different o Stock concentration of ethanol: 2 mM or 2000 M o 9 concentrations in the range of 0 to 200 M is desired (see table below) You make adjust the concentrations in your standard curve as long as 200 M remains the top concentration used. Water to make up the total volume once all other components are accounted for.
Sample table: Well concentration of Ethanol ( M) Volume of buffer (0.15 M) Volume of NAD+ (3 mM) Volume of Enzyme (5000 units/ml) Volume of Ethanol Standard (2 mM) Volume of water Total volume ( l) 200 250 175 250 125 250 100 250 75 250 50 250 10 250 0 250 Remember that 200 M is 0.2 mM If any ethanol volume is less than 2 l plan to make a dilution (10x, 25x, or 50x) of the stock solution to raise the volume in the well above 2 l Background samples: The 0 μ M ethanol concentration can be used for the background for the standard curve since the known ethanol solution is diluted in water. For the unknown samples background, I suggest replacing the enzyme volume with water and keeping all other components of you. This way there will be no reaction that occurs in your sample. Allowing you to consider the buffer, NAD+, any absorbance from your unknown ethanol sample (that isn’t the NADH product of the reaction), and any absorbance that could come from the microplate. o Note: this background would not consider any absorbance of the enzyme.
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