User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2014/03/25

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(Objective)
 
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==Objective==
==Objective==
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Today we'll be determining the molar absorptivities of two different molecules, [http://en.wikipedia.org/wiki/Adenosine adenosine] and [http://en.wikipedia.org/wiki/Inosine inosine]. The data that we generate today will be important when we study [http://en.wikipedia.org/wiki/Adenosine_deaminase adenosine deaminase] (ADA), which converts adenosine to inosine. The difference between these two molecules is that adenosine contains a primary amine whereas inosine contains a carboxy group. Overexpression of this protein causes anemia in humans. A shortage of this protein can lead to severe immuno-defficiency.  
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Today we'll be adding three different concentrations of 30,60, and 90 mM to the 30:1 lysozyme AuNP solutions at three temperatures (4, 22, 37 degrees). Then we will synthesize an additional tube of 30:1 lysozyme AuNP to use as a standard future testing.
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==Procedure==
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''Preparing Samples for Addition of Variables''
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* Pipette 5 mL aliquots into a Falcon tube from the 15 mL samples.
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''Dilution of Variables''
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* The following table shows how we diluted the stock concentrations of various variables.
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* The first graph shows the dilution to 30 mM, the second graph shows 60 mM, and the third graph shows 90 mM.
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* The concentrations of 2,2 bipyridine used are 0.0002316 M, 0.0004544 M, and 0.0006772 M from a stock concentration of 0.0099 M.
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[[Image:3.25.dilution.jac.png|950px|]]
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''Adding Variables to 30:1 Lysozyme-AuNP Solutions''
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# Add 2.5 mL of each variable to each tube.  
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# Place 21 tubes in the oven at 36°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.
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# Place 21 tubes in the freezer at 4°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.
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# Place 21 tubes on the bench at 22°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.
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''Synthesizing 30:1 Lysozyme-AuNP''
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* The following figure describes how we made one tube of 30:1 lysozyme-AuNPs.
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* The sample was heated at 80°C for 4 hours.
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[[Image:3.25.one.tube.png|750px|]]
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Adenosine and inosine have different absorption spectra. We will be observing changes in UV-Vis spectra to determine changes in concentration of both adenosine and inosine. In order to do this, we will need to know the molar absorptivity (ε) of both of these molecules. Just as each molecule has a characteristic absorption at each wavelength, this (per-wavelength) absorption can be quantified by a molar absorptivity. Or ... for a given concentration a molecule will absorb a very specific amount of light at a precise wavelength. A molecule doesn't have just one molar absorptivity; there is a molar absorptivity to describe each wavelength in a molecular absorbance spectrum.
 

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Objective

Today we'll be adding three different concentrations of 30,60, and 90 mM to the 30:1 lysozyme AuNP solutions at three temperatures (4, 22, 37 degrees). Then we will synthesize an additional tube of 30:1 lysozyme AuNP to use as a standard future testing.

Procedure

Preparing Samples for Addition of Variables

  • Pipette 5 mL aliquots into a Falcon tube from the 15 mL samples.


Dilution of Variables

  • The following table shows how we diluted the stock concentrations of various variables.
  • The first graph shows the dilution to 30 mM, the second graph shows 60 mM, and the third graph shows 90 mM.
  • The concentrations of 2,2 bipyridine used are 0.0002316 M, 0.0004544 M, and 0.0006772 M from a stock concentration of 0.0099 M.


Adding Variables to 30:1 Lysozyme-AuNP Solutions

  1. Add 2.5 mL of each variable to each tube.
  2. Place 21 tubes in the oven at 36°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl2, CaCl2, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.
  3. Place 21 tubes in the freezer at 4°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl2, CaCl2, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.
  4. Place 21 tubes on the bench at 22°C: 30 mM, 60 mM, 90 mM of NaCl, KCl, MgCl2, CaCl2, MES, and citric acid, as well as 0.0006772 M, 0.0004544 M, and 0.0002316 M 2,2 bipyridine.


Synthesizing 30:1 Lysozyme-AuNP

  • The following figure describes how we made one tube of 30:1 lysozyme-AuNPs.
  • The sample was heated at 80°C for 4 hours.



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