Continuing to Make Wildtype Asc Hb "Apo"
- Move the dialysis tubing from the overnight dialysis solution to 2L of dH2O.
- Dialyze for about four hours in the dH2O with stirring.
- Move the dialysis tubing from the dH2O into 2L more of dH2O.
- 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
- 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).
- When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer.
- Load 5μL of DNA ladder into the 1st well.
- Load 50μL of the double-digested pQE-80-L-Kan from yesterday into the first wide well.
- Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel.
- Place the gel in Ethidium Bromide Stain for about 30 minutes.
- 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
- The 6-cell unit temperature was lowered to 0.5°C.
- 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.
- 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.
- A spectrum from 800-200nm was taken of the protein in buffer.
- The temperature was increased to 10°C and a spectrum was taken again.
- This was repeated at 20°C, 25°C, 30°C, 40°C, 50°C, 60°C, and 70°C.
- 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.
- A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
- 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.
- 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.
- A spectrum from 800-200nm was taken of the buffer (25mM Tris, 50mM NaCl, pH 8) to be used as a baseline.
- 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.
- 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
|
|