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Objectives:
Objectives:
Design a plan for collecting data to show that all biological systems are affected by complex biotic and abiotic interactions.
Use models to predict and justify that changes in the subcomponents of a biological polymer affect the functionality of the molecule.
Analyze data to identify how molecular interactions affect structure and function.
Things to note in this lab:
Things to note in this lab:
Everything for this lab will be done in the lab notebook.
Make sure you answer the questions for each section of the lab (if called for).
Create your data tables in your notebooks, not on a separate sheet of paper.
We will most likely not be completing part 3 of the lab.
You need to be able to keep track of your syringes and test tubes. MAKE SURE YOU LABEL AND CHECK BEFORE ADDING SOLUTIONS! The reaction will not work and you may have to redo the experiment.
Spectrophotometer
Spectrophotometer
We will be using the spectrophotometer for this lab. It can be quite finicky when using it. We only have 2 working ones so you will need to share the machines. I will show you ahead of time how to use it. If you want to look ahead, you can check out this youtube video.
Part 1 Procedures:
Part 1 Procedures:
Label test tubes and syringes:
2.5 mL syringe "E" for enzyme solution
2.5 mL syringe "P" for product
10 mL syringe "NB" for neutral buffer (pH7)
2.5 mL syringe "S" for substrate (0.1% hydrogen peroxide)
1 test tube "SPNB" for mixture A
1 test tube "ENB" for mixture B
Fill and prepare the labeled syringes and test tubes with the appropriate solutions:
Tube "SPNB"
2 mL of "S"
1 mL of "P"
1 mL of "NB"
Cover "SPNB" with parafilm and slowly invert 2x
Tube "ENB"
1 mL of "E"
3 ml of "NB"
Cover "ENB" with parafilm and slowly invert 2x
Use a disposable pipet and transfer contents in "SPNB" into test tube with "ENB"
Cover "SPNB/ENB" mixture with parafilm and invert slowly 2x
5. Make observations and record data
Use the color palette provided and compare the color of the reaction in the tube.
Record tube # and color in 1 minutes intervals over the course of 5 minutes.
Use the spectrometer to find the wavelength of each test tube during the 5 minutes.
6. Plot the increase in color intensity relative to the color palette over 5 minutes.
7. Calculate the rate of enzymatic activity under these baseline conditions. This is the baseline rate.
8. Set labeled syringes aside for use in the next parts of the lab.
Part 2 Procedures:
Part 2 Procedures:
Record baseline rate from part 1.
Label 12 test tubes (#1-12) following the matching chart in the lab manual or changes that must be made written on the whiteboard.
Tubes # (according to chart or board):
2 mL "S"
1 mL "P"
1 mL "NB" pH7
Tubes # (according to chart or board):
1 mL "E"
3 mL pH3 buffer with "NB" syringe
Repeat step 4 for tubes with their respective pH buffers
Pour to combine reagents from tube with reagents in tube/pH
Observe reaction mixture every minute for 5 minutes by comparing to the color palette. Record absorbance reading as well. Record observations.
Look at pair chart and mix tubes. Repeat step 7 for remaining test tubes.
Calculate rate of reaction for each tube as done in Part 1. Graph pH results (% O2 (mL) per min)
Part 3 Procedures:
Part 3 Procedures:
Plan investigation in lab notebook. Include:
Question or testable hypothesis
Background info (brief)
Describe experiment design: controls, variables, observations
Describe possible results and how will interpret data
List materials and methods
Note potential safety issues
Get plan approved.
Document step-by-step procedure including any changes or mishaps. Record volumes, weights and any measurements used.
Record results.
Analyze results.
Make conclusion(s).
Indicate limitations and possible future studies.
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