User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/13

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Continuing to Make Wildtype Asc Hb "Apo"

  1. Move the dialysis tubing from the overnight dialysis solution to 2L of dH2O.
  2. Dialyze for about four hours in the dH2O with stirring.
  3. Move the dialysis tubing from the dH2O into 2L more of dH2O.
  4. Dialyze overnight in the dH2O with stirring.

Reconstitute Asc Hb with Manganese

  • The stir plate was turned off at the beginning of the day, so the protein/Mn-porphyrin mixture could sit undisturbed.

Continuing The Wizard® SV Gel and PCR Clean-Up System

The procedure from Promega was followed starting where I left off yesterday. The DNA purification was done by centrifugation. The following deviations from the procedure were done:

  • The tabletop centrifuge we have only has a max speed of 13200rpm, so this speed was used instead of 14000rpm (which may affect yield).
  • The step to evaporate the additional ethanol with no cover on the centrifuge was done in a mini centrifuge for 1.5min (only has a max speed of about 6000rpm).

Running a DNA Gel of Yesterday's Double Digest pQE-80-L-Kan

  1. Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells).
  2. When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer.
  3. Load 5μL of DNA ladder into the 1st well.
  4. Load 50μL of the double-digested pQE-80-L-Kan from yesterday into the first wide well.
  5. Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel.
  6. Place the gel in Ethidium Bromide Stain for about 30 minutes.
  7. View under a UV light.
    • Be careful when working with ethidium bromide and UV light.

The DNA will not be gel purified. It seems I'm having the same trouble I did before because there are two fragments and their sizes are approximately between 1.5-2kb and between 3-4kb.

Effect of Temperature on wildtype Asc Hb Spectra

  1. The 6-cell unit temperature was lowered to 0.5°C.
  2. 850μL of buffer was mixed with 150μL of the concentrated wildtype Asc Hb (previously purified and concentrated by User:Matt Hartings in a quartz cuvette.
  3. The cuvette was placed in the spectrophotometer and given time to cool.
    • There was a problem with "fogging" on the sides of the cuvette with the lower temperatures, so the cuvette was removed and wiped with a Kimwipe before the readings at lower temperatures were taken.
  4. A spectrum from 800-200nm was taken of the protein in buffer.
  5. The temperature was increased to 10°C and a spectrum was taken again.
  6. This was repeated at 20°C, 25°C, 30°C, 40°C, 50°C, 60°C, and 70°C.
  7. A spectrum from 800-200nm was taken of just the buffer (25mM Tris, 50mM NaCl, pH 8) at 25°C to be used as a baseline.

Effect of Guanidine-HCl on Wildtype Asc Hb Spectra

  • 5M Guanidine-HCl was prepared on 06/08/12 by dissolving 4.78g of Guanidine-HCl in dH2O. The final volume of the solution was brought to 10mL with dH2O in a volumetric flask.
  • All of these spectra readings were taken at 25°C.
  1. A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
  2. Spectra of the following mixtures were taken from 200-800nm. For each mixture, the cuvette was covered with parafilm when all components were in it and inverted a few times to thoroughly mix.
  3. For each mixture about 200μL of it was transferred to a fluorescence cuvette, and the cuvette was placed in a fluorometer. The mixture was excited at 280nm, and a scan was taken from 300-450nm.
Mixtures with Various Concentrations of Guanidine-HCl
Amount of Buffer Amount of 5M Guanidine-HCl Final Concentration of Guanidine-HCl in Cuvette Amount of Wildtype Asc Hb
850 μL 0 μL 0M 150 μL
830 μL 20 μL 0.1M 150 μL
800 μL 50 μL 0.25M 150 μL
750 μL 100 μL 0.5M 150 μL
700 μL 150 μL 0.75M 150 μL
650 μL 200 μL 1M 150 μL
550 μL 300 μL 1.5M 150 μL
450 μL 400 μL 2M 150 μL
350 μL 500 μL 2.5M 150 μL
250 μL 600 μL 3M 150 μL

Effect of Urea on Wildtype Asc Hb Spectra

  • 5M Urea was prepared on 06/08/12 by dissolving 3.00g of Urea in dH2O. The final volume of the solution was brought to 10mL with dH2O in a volumetric flask.
  • All of these spectra readings were taken at 25°C.
  1. A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
  2. Spectra of the following mixtures were taken from 200-800nm. For each mixture, the cuvette was covered with parafilm when all components were in it and inverted a few times to thoroughly mix.
  3. For each mixture about 200μL of it was transferred to a fluorescence cuvette, and the cuvette was placed in a fluorometer. The mixture was excited at 280nm, and a scan was taken from 300-450nm.
Mixtures with Various Concentrations of Urea
Amount of Buffer Amount of 5M Urea Final Concentration of Urea in Cuvette Amount of Wildtype Asc Hb
850 μL 0 μL 0M 150 μL
830 μL 20 μL 0.1M 150 μL
800 μL 50 μL 0.25M 150 μL
750 μL 100 μL 0.5M 150 μL
700 μL 150 μL 0.75M 150 μL
650 μL 200 μL 1M 150 μL
550 μL 300 μL 1.5M 150 μL
450 μL 400 μL 2M 150 μL
350 μL 500 μL 2.5M 150 μL
250 μL 600 μL 3M 150 μL




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