User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/30

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Tasklist

We will continue the rotation that we started last week. All groups will set up dialysis.
1 Group will work on GPC.
The other two will either retry Bradford, create a Ca2+ calibration, or a Cl- calibration.

  1. Dialysis
    • Prepare 10 mL 0.5 g/L Lysozyme
    • Prepare 2 mL 50 mM CaCl2 (if you read ahead, you can use your CaCl2 calibration stock)
    • Prepare 2 mL 500 μM CaCl2 (if you read ahead, you can use a CaCl2 calibration dilution)
    • Prepare 2 mL 50 mM NaCl (if you read ahead, you can use your NaCl calibration stock)
    • Prepare 1 mL 0.25 mM HCl using your 2.5 mM stored stock
    • Cut a piece of 3500 MWCO flat dialysis tubing, wet it, and insert it in the 5-well dialysis chamber
    • Clamp the dialysis chamber shut
    • Fill one side of each well with 1 mL of your 0.5 g/L Lysozyme solution
      • Transfer 1 mL to a plastic test tube using a pippetter, then
      • Carefully fill each well with with a glass Pasteur pipette
      • You may need to tip the chamber on an angle to reduce water surface tension
    • Fill the other side of the well with one of the following solutions
      • HPLC water, 0.25 mM HCl, 50 mM CaCl2, 500 μM CaCl2, or 50 mM NaCl
      • Each well has a different solution, which will be tested tomorrow
    • Insert top screws to prevent leaking/evaporation
  2. GPC
    • Prepare 500 mL 0.5M HAc / 0.3M Na2SO4
    • Prepare 10 mL 1 g/L Lysozyme using the HAc/Na2SO4 stock
    • Using your 1 g/L Lysozyme & HAc/Na2SO4 stock, prepare 5 mL 0.5 g/L & 5 mL 0.1 g/L Lysozyme
    • Filter your remaining HAc/Na2SO4 stock through 0.2 μM membrane filter into clean & dry filter flask
    • Run GPC (I will instruct you)
  3. Ca2+ calibration curve
    • Prepare 25 mL 50 mM CaCl2 using HPLC water
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
    • Measure Ca2+ using ISE
    • Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg2+ should, Na+ should not)
    • Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
  4. Cl- calibration curve
    • Prepare 25 mL 50 mM NaCl using HPLC water
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
    • Measure Cl- using ISE
    • Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
    • Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.