User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/25

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(DATA/Calculations)
Current revision (17:30, 27 November 2012) (view source)
(Procedure)
 
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#Sonicate the cells using sonication instrumentation. place the sonication device inside each of vials containing the cells for 30 seconds followed placing the vial in an ice bath for 30 seconds. repeat this sequence three times. This will shock and open up the cells.  
#Sonicate the cells using sonication instrumentation. place the sonication device inside each of vials containing the cells for 30 seconds followed placing the vial in an ice bath for 30 seconds. repeat this sequence three times. This will shock and open up the cells.  
#centrifuge the cells at 18000 rpm and 4°C for 2 hours. This will separate the organelles of the cells from the proteins we want.
#centrifuge the cells at 18000 rpm and 4°C for 2 hours. This will separate the organelles of the cells from the proteins we want.
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#Meanwhile, filter the binding buffer and elution buffer in order to purify them using a vacuum system of filtration and filter paper with a membrane filter in which the holes measure 450nm.  
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#Meanwhile, filter the [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19| binding buffer]] and [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19| elution buffer]] in order to purify them using a vacuum system of filtration and filter paper with a membrane filter in which the holes measure 450nm.  
#Once the centrifuging is completed. Use the same vacuum filter system to separate the proteins from everything else in the vials (organelles, buffer, etc.)
#Once the centrifuging is completed. Use the same vacuum filter system to separate the proteins from everything else in the vials (organelles, buffer, etc.)
# The protein was collected in falcon vials and refrigerated.  
# The protein was collected in falcon vials and refrigerated.  
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1mol/L x .025= .025 mol
1mol/L x .025= .025 mol
.025 mol x 121.14 g/mol = 3.0285 g of tris  
.025 mol x 121.14 g/mol = 3.0285 g of tris  
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:01, 7 October 2012 (EDT)''':you need to note that this is a 1.0 M Tris stock.
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To get the necessary concentrations of tris into the gold/BSA solutions, the M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub> formula was used to calculate the volume of tris needed. From there, the value of the volume of tris was subtracted from 1 in order to determine the amount of water that would be added to make the desired concentration.
To get the necessary concentrations of tris into the gold/BSA solutions, the M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub> formula was used to calculate the volume of tris needed. From there, the value of the volume of tris was subtracted from 1 in order to determine the amount of water that would be added to make the desired concentration.
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link for excel [[Image:TrisvaryingmolarUVVis.xlsx| Spreadsheets]]
link for excel [[Image:TrisvaryingmolarUVVis.xlsx| Spreadsheets]]
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:02, 7 October 2012 (EDT)''':what is hte difference between trials one and two? what am i looking for?
==Conclusions==
==Conclusions==
* Todays steps were necessary in order to separate out the proteins from the cells and collect them.  
* Todays steps were necessary in order to separate out the proteins from the cells and collect them.  
* sample 1 cells are still being tested separately due to the original discrepancy. filtration, sonication, all were performed separately for the main sample and sample 1.  
* sample 1 cells are still being tested separately due to the original discrepancy. filtration, sonication, all were performed separately for the main sample and sample 1.  
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* In repeating the same experiment as...... we conducted a uv vis.....
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* In repeating the same experiment as the previous class using varying concentrations of the tris buffer with the AuNP/BSa solution, no difference could be seen between the trials except for 5mM in the second trial. Either another trial needs to be run at this concentration, or a blank needs to be run to see if it is accountable in any way for the jump in absorbance.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:05, 7 October 2012 (EDT)''': you have not provided enough information to know why a blank would help. also why didn't you run a blank? a blank should always be run. when do you plan to assess this experiment again? and will you test both conclusions? and oyu have not provided enough information to explain your conclusions? nothing drastic is different but without knowing what changed between the two you cannot draw conclusions about them
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Separate Proteins

Goals

  • Separate the proteins from the cells

Procedure

  • Separate Proteins
  1. Remove the cells from the -80°C Freezer and place in a water bath to unfreeze the cells and bring them to room temperature.
  2. Sonicate the cells using sonication instrumentation. place the sonication device inside each of vials containing the cells for 30 seconds followed placing the vial in an ice bath for 30 seconds. repeat this sequence three times. This will shock and open up the cells.
  3. centrifuge the cells at 18000 rpm and 4°C for 2 hours. This will separate the organelles of the cells from the proteins we want.
  4. Meanwhile, filter the binding buffer and elution buffer in order to purify them using a vacuum system of filtration and filter paper with a membrane filter in which the holes measure 450nm.
  5. Once the centrifuging is completed. Use the same vacuum filter system to separate the proteins from everything else in the vials (organelles, buffer, etc.)
  6. The protein was collected in falcon vials and refrigerated.
  • UV-Vis
  1. New concentrations of the Tris buffer were added to the Gold/BSA solution. These solutions did not express any fibers, it was just purple solution. The UV-Vis was run twice on the samples with an hour in between each run.

DATA/Calculations

  • Uv-Vis:
Tris Buffer concentration amount of water (mL) amount of tris (mL)
.05 mM0.99380.0625
.5 mM0.98750.0125
5 mM0.9750.025
50 mM0.950.05
100 mM0.90.1
200 mM0.80.2
500 mM0.50.5
1 M01

to make a 25mL stock solution of the tris buffer at a pH of 10 1mol/L x .025= .025 mol .025 mol x 121.14 g/mol = 3.0285 g of tris


  • Abigail E. Miller 11:01, 7 October 2012 (EDT):you need to note that this is a 1.0 M Tris stock.


To get the necessary concentrations of tris into the gold/BSA solutions, the M1V1=M2V2 formula was used to calculate the volume of tris needed. From there, the value of the volume of tris was subtracted from 1 in order to determine the amount of water that would be added to make the desired concentration.


Graphs of run 1 and 2

Tris varying concentrations at pH 10

link for excel Image:TrisvaryingmolarUVVis.xlsx

  • Abigail E. Miller 11:02, 7 October 2012 (EDT):what is hte difference between trials one and two? what am i looking for?

Conclusions

  • Todays steps were necessary in order to separate out the proteins from the cells and collect them.
  • sample 1 cells are still being tested separately due to the original discrepancy. filtration, sonication, all were performed separately for the main sample and sample 1.
  • In repeating the same experiment as the previous class using varying concentrations of the tris buffer with the AuNP/BSa solution, no difference could be seen between the trials except for 5mM in the second trial. Either another trial needs to be run at this concentration, or a blank needs to be run to see if it is accountable in any way for the jump in absorbance.
  • Abigail E. Miller 11:05, 7 October 2012 (EDT): you have not provided enough information to know why a blank would help. also why didn't you run a blank? a blank should always be run. when do you plan to assess this experiment again? and will you test both conclusions? and oyu have not provided enough information to explain your conclusions? nothing drastic is different but without knowing what changed between the two you cannot draw conclusions about them


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