User:Catherine Koenigsknecht/Notebook/Experimental Biological Chemistry/2013/02/06

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*Phosphate buffer was used as a blank and was used to create the baseline.  
*Phosphate buffer was used as a blank and was used to create the baseline.  
 +
*Kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table below).
 +
**The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
 +
*5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
*5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
 +
**150 uM adenosine stock solution was used; (stock) 150uL 5mM stock + 850 uL buffer =750 uM ->use .6mL new stock for ADA assay
 +
**200 uM adenosine stock solution was used: (stock) 200 uL 5mM stock+800 uL buffer = 1000uM ->use 0.6mL new stock for ADA assay
**This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
**This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
***[[Image:Screen_Shot_2013-02-05_at_9.01.20_PM.png]]
***[[Image:Screen_Shot_2013-02-05_at_9.01.20_PM.png]]
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==Data==
==Data==
-
* Add data and results here...
 
-
==Notes==
+
{| {{table}}
-
This area is for any observations or conclusions that you would like to note.
+
| align="center" style="background:#f0f0f0;"|'''Concentration of Adenosine (uM)'''
 +
| align="center" style="background:#f0f0f0;"|'''Average Velocity over 30s (nm/sec)'''
 +
|-
 +
| 200||0.00021
 +
|-
 +
| 100||0.000183333
 +
|-
 +
| 40||0.00012
 +
|-
 +
| 16||0.0001
 +
|-
 +
| 6.4||0.000047
 +
|-
 +
| 2.56||0.000033
 +
|}
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''1/[adenosine] (1/uM)'''
 +
| align="center" style="background:#f0f0f0;"|'''1/[velocity] (sec/nm)'''
 +
|-
 +
| 0.005||4761.904762
 +
|-
 +
| 0.01||5454.545455
 +
|-
 +
| 0.025||8333.333333
 +
|-
 +
| 0.0625||10000
 +
|-
 +
| 0.15625||21276.59574
 +
|-
 +
| 0.390625||30303.0303
 +
|}
 +
 +
 +
*Lineweaver Burke Plot:
 +
[[Image:ConcentrationvsVelocity-1.png]]
 +
 +
*Concentration Adenosine vs Velocity:
 +
[[Image:LineweaverBurkePlot-1.png]]
 +
 +
{| {{table}}
 +
| -1/Vmax||6163.1
 +
|-
 +
| '''Vmax'''||'''0.000162256 nm/sec'''
 +
|-
 +
| -1/Km||-0.093
 +
|-
 +
| '''Km'''||'''10.75 uM'''
 +
|}
 +
 +
==Notes==
 +
Concentration recalculations were taken from Dhea Patel
 +
Deleted concentration 2.56 uM point from graphs
Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.
Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.

Revision as of 12:59, 12 February 2013

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Entry title

Objective

Learn how to maintain an OpenWetWare Notebook.

Description

  1. UVProbe was opened.
    1. Window > 1. Kinetics
    2. Methods Icon
      1. Wavelength: 265nm
      2. Duration: 300 seconds (5minutes)
      3. OK
  2. Shimadzu CPS-Controller was set to 25°C.
    1. wait for the temperature to raise to 25°C
  3. place the sample in the cell and click start.
  • Phosphate buffer was used as a blank and was used to create the baseline.
  • Kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table below).
    • The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).


  • 5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
    • 150 uM adenosine stock solution was used; (stock) 150uL 5mM stock + 850 uL buffer =750 uM ->use .6mL new stock for ADA assay
    • 200 uM adenosine stock solution was used: (stock) 200 uL 5mM stock+800 uL buffer = 1000uM ->use 0.6mL new stock for ADA assay
    • This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
      • Image:Screen_Shot_2013-02-05_at_9.01.20_PM.png
    • This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
      • Image:Screen_Shot_2013-02-05_at_9.01.33_PM.png

Data

Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
2000.00021
1000.000183333
400.00012
160.0001
6.40.000047
2.560.000033


1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.0054761.904762
0.015454.545455
0.0258333.333333
0.062510000
0.1562521276.59574
0.39062530303.0303


  • Lineweaver Burke Plot:

Image:ConcentrationvsVelocity-1.png

  • Concentration Adenosine vs Velocity:

Image:LineweaverBurkePlot-1.png

-1/Vmax6163.1
Vmax0.000162256 nm/sec
-1/Km-0.093
Km10.75 uM

Notes

Concentration recalculations were taken from Dhea Patel Deleted concentration 2.56 uM point from graphs

Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.



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