Objectives
- Extract ADA from E. Coli
- Obtain two sets of UV/Vis spectra for Au/BSA solutions with varying concentrations of Tris, to see how absorbance changes over time
Procedure
- Protein extraction
- Cells were removed from freezer and warmed to room temperature in a water bath.
- Cells were sonicated to break membranes and allow ADA and the other contents of the cell into the broth
- Abigail E. Miller 11:42, 7 October 2012 (EDT):you are not in borth, the cells were resuspended in binding buffer before freezing.
- Each vial was placed in an ice bath for 30 seconds
- Sonication device was placed in each vial for 30 seconds
- This was repeated 3 times for each vial.
- Cells were centrifuged at 18000rpm and 4°C for 2 hours to seperate ADA from E. Coli
- Abigail E. Miller 11:42, 7 October 2012 (EDT):it is to remove large particlate and matter. e. coli is destroyed by sonication leaving you with all the pieces.
- Binding and elution buffers were purified via vacuum filtration and a 450nm membrane filter.
- A buchner funnel was attached to a flask, along with a vacuum pump.
- A 450nm membrane was put in the buchner funnel
- The binding buffer was filtered first, so that imidazole left behind by the elution buffer would not contaminate it.
- Abigail E. Miller 11:42, 7 October 2012 (EDT):it is so hte higher conncetration imidizole solution, the elution buffer, does not increase the in=midizole concnetration of the binding buffer
- Vacuum filtration was used to seperate proteins from cells. ADA was collected in falcon vials and refridgerated.
- Abigail E. Miller 11:42, 7 October 2012 (EDT):more than just ADA is left, hence the chromatography.
- Solutions of Au/BSA at a mole ratio of 70 (at which fibers are not produced) with Tris at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM, and 1M went through two rounds each of UV/Vis.
- Abigail E. Miller 11:42, 7 October 2012 (EDT):what is different between each spectrum? time? addition of Tris?
- Stock solution for Tris was made
- 1mol/L *.025L = .025mol Tris needed
- .025mol * 121.14g/mol = 3.0285g Tris weighed
- The equation M1V1=M2V2 was used to calculate the amount of stock and water needed in the 100mM, 200mM, 500mM and 1M tubes. A serial dilution was done from 500mM to .05mM to fill the rest of the tubes. 1mL from each tube was moved to the next with a pipette.
Data
Amount of Tris stock and water in each tube
| Tris Buffer concentration
|
amount of water (mL)
|
amount of tris (mL)
|
| .05 mM |
0.9938 |
0.0625
|
| .5 mM |
0.9875 |
0.0125
|
| 5 mM |
0.975 |
0.025
|
| 50 mM |
0.95 |
0.05
|
| 100 mM |
0.9 |
0.1
|
| 200 mM |
0.8 |
0.2
|
| 500 mM |
0.5 |
0.5
|
| 1 M |
0 |
1
|
Data and Conclusions
Results of other team:
- No significant difference is seen between our two UV/Vis trials, except for 5mM in the second trial, which has a much higher absorbency peak. More trials should be run to see if this is random error.
- Abigail E. Miller 11:42, 7 October 2012 (EDT):will you test this?
- Opposite trends can be seen between the two teams' trials. In ours, lower molarities of Tris resulted in higher absorbance, while for the other team, lower absorbance for lower molarities of Tris could be seen. Their absorbance values were also much higher.
- Abigail E. Miller 11:42, 7 October 2012 (EDT):what does this mean? why might it matter? why might you have differing results from the previous group? and what group are you referring too?
- Abigail E. Miller 11:42, 7 October 2012 (EDT):note: team implies competition, group is not so aggressive. while both terms are likely appropriate, this is not a competition. :)
|
Project name
