User:Michael F. Nagle/Notebook/Chem 571/2012/09/25: Difference between revisions

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##Cells were centrifuged at 18000rpm and 4°C for 2 hours to seperate ADA from E. Coli
##Cells were centrifuged at 18000rpm and 4°C for 2 hours to seperate ADA from E. Coli
## Binding and elution buffers were purified via vacuum filtration and a 450nm membrane filter.
## 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.
## Vacuum filtration was used to seperate proteins from cells. ADA was collected in falcon vials and refridgerated.
## Vacuum filtration was used to seperate proteins from cells. ADA was collected in falcon vials and refridgerated.
#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.
#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.
##Stock solution for Tris was made  
##Stock solution for Tris was made  

Revision as of 14:54, 6 October 2012

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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

  1. Protein extraction
    1. Cells were removed from freezer and warmed to room temperature in a water bath.
    2. Cells were sonicated to break membranes and allow ADA and the other contents of the cell into the broth
      1. Each vial was placed in an ice bath for 30 seconds
      2. Sonication device was placed in each vial for 30 seconds
      3. This was repeated 3 times for each vial.
    3. Cells were centrifuged at 18000rpm and 4°C for 2 hours to seperate ADA from E. Coli
    4. Binding and elution buffers were purified via vacuum filtration and a 450nm membrane filter.
      1. A buchner funnel was attached to a flask, along with a vacuum pump.
      2. A 450nm membrane was put in the buchner funnel
      3. The binding buffer was filtered first, so that imidazole left behind by the elution buffer would not contaminate it.
    5. Vacuum filtration was used to seperate proteins from cells. ADA was collected in falcon vials and refridgerated.
  2. 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.
    1. Stock solution for Tris was made
      1. 1mol/L *.025L = .025mol Tris needed
      2. .025mol * 121.14g/mol = 3.0285g Tris weighed
    2. 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

  • No significant difference is seen between 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.


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