User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/10/01: Difference between revisions

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==Entry title==
==Tasklist==
* Insert content here...
# GPC for one more group
# Finish Ca<sup>2+</sup> and Cl<sup>-</sup> calibration curve & interferences
# Analyze Dialysis Solutions
# Try week long dialysis using different CaCl<sub>2</sub> solutions or 20,000 MWCO tubing?
 
==Analysis of Dialysis Solutions==
* Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
**Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
**Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
**PS cuvettes, measuring 400 - 800 nm
**Don't forget to run a blank with just Bradford & buffer
**Don't forget to run your undialyzed Lysozyme stock
* Transfer remainder of each cell into 20 mL extraction vials
* Measure Ca<sup>2+</sup> using ISE
* Transfer 100 μL to a small volume UV cuvette & measure UV absorption
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
**Measure entire 200 - 800 nm range
* Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
**excitation at 280 nm
* Transfer 500 - 700 μL to a 15 mL Falcon tube.  Add 2 - 3 times volume of water & measure pH
**You want to add minimum amount of water
**You typically need 1.6 - 2 mL in falcon tube to cover pH probe
**So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water)
**Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water)




Revision as of 08:07, 1 October 2014

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Tasklist

  1. GPC for one more group
  2. Finish Ca2+ and Cl- calibration curve & interferences
  3. Analyze Dialysis Solutions
  4. Try week long dialysis using different CaCl2 solutions or 20,000 MWCO tubing?

Analysis of Dialysis Solutions

  • Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm
    • Don't forget to run a blank with just Bradford & buffer
    • Don't forget to run your undialyzed Lysozyme stock
  • Transfer remainder of each cell into 20 mL extraction vials
  • Measure Ca2+ using ISE
  • Transfer 100 μL to a small volume UV cuvette & measure UV absorption
    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • Measure entire 200 - 800 nm range
  • Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence
    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • excitation at 280 nm
  • Transfer 500 - 700 μL to a 15 mL Falcon tube. Add 2 - 3 times volume of water & measure pH
    • You want to add minimum amount of water
    • You typically need 1.6 - 2 mL in falcon tube to cover pH probe
    • So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water)
    • Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water)



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