Effect of Urea on Wildtype Asc Hb Reconstituted with Nickel 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 Reconstituted with Nickel
|
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 Reconstituted with Manganese 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 Reconstituted with Manganese
|
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 Guanidine-HCl on Wildtype Asc Hb Reconstituted with Manganese 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 Reconstituted with Manganese
|
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 Guanidine-HCl on Wildtype Asc Hb Reconstituted with Nickel 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 Reconstituted with Nickel
|
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
|
Transformations
This was done with commercially competent BL21 and NovaBlue E.coli. Each strain was attempted to be transformed with both wildtype and triple mutant Asc Hb (M8S/M33S/M103S) ligations with pQE-80-L-Kan from 7/11/12:
- Place plastic culture tubes on ice for about 30 min.
- Place DNA for transformation on ice.
- After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
- Add 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
- Add cells (25uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
- Put tube back in ice for 4 minutes.
- Heat shock at 42°C for 80 seconds.
- Add 100uL of SOC media.
- Shake at 37°C for 1 hour.
- Plate 50uL of culture media on LB/amp plates (prepared fresh today: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, add 250μL of 100mg/mL ampicillin).
- Incubate inverted overnight at 37°C.
- Note 8/3/12: no growth overnight or throughout the day, will leave at room temperature over the weekend to see if the colonies are slow growing
|