Objective
For this lab session, our group (GRP 3) will be doing oceanography and prepping samples with our assigned protease: trypsin. After completing oceanography readings, we will also redo stock solutions to have gold fibers instead of nanoparticles, and prepare more trypsin samples for later use.
Procedure
- Centrifuged eppindorf tubes containing gold fibers.
- Removed water with plastic pipette.
- Prepare the sample that goes in the 3mL cuvette that will be used for ocean optics.
- The sample must contain
- 1mL of gold fibers
- 0.3mL of buffer
- 0.0573mL of trypsin, calculations are below
- The rest of the volume was filled with water: 1 - (1mL fibers + 0.3mL buffer + 0.0573mL trypsin)= 1.643mL water.
- The trypsin was added last, at the second reading such that second reading becomes time 0.
- We ran our sample for 2hrs30mins.
- Readings were taken every 2 minutes and uploaded directly to DropBox for later analysis.
Calculations for volume of trypsin:
C1·V1 = C2·V2,
where C1 = 52.36µM
C2 = 1µM
V2 = 3mL
V1 = 57.3µL
- Preparation of stock solution containing fibers for analysis
- We followed Dr. Hartings' modified protocol for making the fiber samples
- Preparation of trypsin samples.
- Weigh 15 eppendorf tubes and record their mass.
- To each tube, add approximately 1mg of trypsin, weigh and record.
- Calculate the concentration in each tube if 1mL of liquid were added.
Calculations to
Data
Table 1. Measurements taken while making the trypsin sample tubes
Number of Eppi Tube
|
Mass of Eppi Tube (g)
|
Mass of Trypsin (g)
|
Concentration (µM)
|
1 |
1.02691 |
0.00108 |
46.35
|
2 |
1.02622 |
0.00148 |
63.52
|
3 |
1.0224 |
0.00117 |
50.21
|
4 |
1.01944 |
0.00169 |
72.33
|
5 |
1.02922 |
0.00181 |
77.68
|
6 |
1.02625 |
0.00128 |
54.93
|
7 |
1.01909 |
0.00165 |
70.82
|
8 |
1.02036 |
0.00182 |
78.11
|
9 |
1.01922 |
0.00128 |
54.93
|
10 |
1.02024 |
0.00122 |
52.36
|
11 |
1.02097 |
0.00154 |
66.09
|
12 |
1.02422 |
0.00175 |
75.11
|
13 |
1.02669 |
0.00124 |
53.22
|
14 |
1.02936 |
0.00148 |
63.52
|
15 |
1.02923 |
0.00155 |
66.52
|
Table 2. Raw data for the graph following it. The graph is a Concentration vs. Absorbance of the class's compiled UV/Vis data at 280 nm.
Group
|
Concentration
|
A(280nm)
|
Group 1 |
2 |
0.052
|
|
5 |
0.16
|
|
8 |
0.239
|
|
10 |
0.292
|
|
15 |
0.444
|
Group 3 |
0.7875 |
0.026
|
|
2.625 |
0.091
|
|
5.25 |
0.186
|
|
7.5 |
0.252
|
|
15 |
0.502
|
Group 4 |
0.965 |
0.032
|
|
1.93 |
0.069
|
|
3.86 |
0.123
|
|
7.225 |
0.261
|
|
15.445 |
0.517
|
Group 5 |
1.56 |
0.052
|
|
3.13 |
0.108
|
|
6.25 |
0.221
|
|
12.5 |
0.441
|
|
15 |
0.54
|
Graph 1.
Graph 2.
Graph 3.
|