User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/02

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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.
  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 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.
  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 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.
  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 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.
  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 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:

  1. Place plastic culture tubes on ice for about 30 min.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (25uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour.
  10. 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).
  11. 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