User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/30: Difference between revisions

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*Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg<sup>2+</sup> should, Na<sup>+</sup> should not)
*Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg<sup>2+</sup> should, Na<sup>+</sup> should not)
*Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
*Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Substance'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
|-
| Tap water || 51.3
|-
| HPLC water || -23.0
|-
| DI water ||-51.5
|-
|}
#Cl<sup>-</sup> calibration curve
#Cl<sup>-</sup> calibration curve
#*Prepare 25 mL 50 mM NaCl using HPLC water
#*Prepare 25 mL 50 mM NaCl using HPLC water
Line 56: Line 67:
#*Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
#*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.
#*Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Substance'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
|-
| Tap water || 65.5
|-
| HPLC water || 163.5
|-
| DI water ||158.8
|-
|}
===Dyalisis===
===Dyalisis===
#Dialysis
#Dialysis

Revision as of 12:04, 30 September 2014

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Tasks for September 30

  • To run dyalisis
  • To prepare calibration curves Ca2+ and Cl- calibration curves

Making up calibration curves for Ca2+ and Cl-

Procedure followed as described by Dr. Fox

  1. Ca2+ calibration curve
    • Prepare 25 mL 50 mM CaCl2 using HPLC water
      • Added 0.13876g CaCl2 to a 25 mL volumetric flask and made up to the mark with HPLC
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
    • Measure Ca2+ using ISE
CaCl2 concentration mV measurement (mV)
5μM -44.6
50μM 8.2
500μM 39.5
5mM 63.5
50mM 86.6
  • 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.
Substance mV measurement (mV)
Tap water 51.3
HPLC water -23.0
DI water -51.5
  1. Cl- calibration curve
    • Prepare 25 mL 50 mM NaCl using HPLC water
      • Added 0.07091 g NaCl to a 25 mL volumetric flask and made up to the mark with HPLC
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
    • Measure Cl- using ISE
NaCl concentration mV measurement (mV)
5μM 146.5
50μM 129.6
500μM 81.3
5mM 27.5
50mM -23.9
    • 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.
Substance mV measurement (mV)
Tap water 65.5
HPLC water 163.5
DI water 158.8

Dyalisis

  1. Dialysis
    • Prepare 10 mL 0.5 g/L Lysozyme
      • Added 0.0050 g to 10mL volumetric flask and made up to mark with HPLC
    • 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
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