Tasklist
- GPC for one more group
- Finish Ca2+ and Cl- calibration curve & interferences
- Analyze Dialysis Solutions
- 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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