User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/27

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Running a DNA Gel

  1. A 1.2% agarose gel was made by combining 0.3g agarose with 25mL TAE buffer, microwaving the mixture until boiling, pouring the mixture into gel chamber, and placing a comb for wells.
  2. The first well was loaded with 5μL of pre-mixed ladder.
  3. Then a small well and a large well were skipped.
  4. The second well loaded was loaded with 5μL of yesterday's room temperature ligation with wildtype Asc Hb into pQE-80-L-Kan mixed with 1μL of 6x loading dye.
  5. The third well loaded was loaded with 5μL of yesterday's room temperature ligation with M8S/M33S/M103S Asc Hb into pQE-80-L-Kan mixed with 1μL of 6x loading dye.
  6. The fourth well loaded was loaded with 5μL of last night's overnight ligation (16°C) with wildtype Asc Hb into pQE-80-L-Kan mixed with 1μL of 6x loading dye.
  7. The fifth well loaded was loaded with 5μL of last night's overnight ligation (16°C) with M8S/M33S/M103S Asc Hb into pQE-80-L-Kan mixed with 1μL of 6x loading dye.

Image: Photo_(20).JPG

Again it does not look like a ligation occured. There does seem to be some DNA still in the well for well loaded with 5μL of last night's overnight ligation (16°C) with wildtype Asc Hb into pQE-80-L-Kan mixed with 1μL of 6x loading dye.

Making Media

Autoclave all of these on a liquid cycle:

  • 100mL LB: 2.5g LB (w/o NaCl) + 1g NaCl
  • 250mL LB (for plates): 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar
    • Add 250μL of 100mg/mL ampicillin after the media has been autoclaved and has cooled to about 60°C, then pour plates

Bradford Protein Assay

Standards were set-up in the following way in plastic cuvettes:

Standard Amount of Bradford Dye Amount of 14.6μg/mL BSA Amount of Distilled Water Final Concentration in Cuvette
Blank 200 μL 0 μL 800 μL 0 μg/mL
Standard 1 200 μL 50 μL 750 μL 0.73 μg/mL
Standard 2 200 μL 100 μL 700 μL 1.46 μg/mL
Standard 3 200μL 250μL 550μL 3.65μg/mL
Standard 4 200μL 400μL 400μL 5.84μg/mL
Standard 5 200μL 550μL 250μL 8.03μg/mL
Standard 6 200μL 700μL 100μL 10.22μg/mL
  • The BSA was diluted from 1.46mg/mL to 14.6μg/mL by adding 5μL of 1.46mg/mL BSA to 495μL of distilled water.
  • Then, when more BSA was needed, 10μL of 1.46mg/mL BSA was added to 990μL of distilled water.
  • And when more was needed one more time, 10μL of 1.46mg/mL BSA was added to 990μL of distilled water.
  1. Spectra from 200nm-800nm was taken of each of the standards and Blank.
  2. The wavelength recorded at 595nm was used to create a standard curve for protein concentration.
  3. 200μL of Bradford dye was mixed with various dilutions and volumes of the wildtype Asc Hb and dH2O to a final volume of 1mL.
  4. Spectra from 200nm-800nm was taken of the various concentrations of wildtype Asc Hb.
  5. The wavelength recorded at 595nm was used to calculate the protein concentration of the wildtype Asc Hb.

Bradford Protein Assay Results

The absorbance from the blank (800μL distilled water with 200μL Bradford dye) was subtracted from the absorbances of the standards at 595nm to create the standard curve to determine protein concentration. The absorbance of the blank was 0.618.

Standard Concentration Absorbance Corrected Absorbance
Standard 1 0.73 μg/mL 0.646 0.028
Standard 2 1.46 μg/mL 0.713 0.095
Standard 3 3.65μg/mL 0.876 0.191
Standard 4 5.84μg/mL 0.948 0.33
Standard 5 8.03μg/mL 1.082 0.464
Standard 6 10.22μg/mL 1.142 0.524

Image:Bradford_standard_062712.JPG


  • The equation of this line was determined by Excel to be (forced through the origin) y=0.0542x, where y is the absorbance and x is the concentration.
  • The absorbance of the blank was also subtracted from the wildtype Asc Hb/Bradford absorbances at 595nm.
  • There was a concern that the wildtype Asc Hb itself would have an effect on the absorbance taken at 595nm, but it turns out the absorbance was negligible. The absorbance of concentrated wildtype Asc Hb in a plastic cuvette at 595nm was 0.09, and the corrected absorbance (subtracting the absorbance of water in a plastic cuvette at 595nm of 0.051) is 0.039. This is a very small effect, and the effect is negligible when the protein is diluted 20 times or more.
  • The concentration of the protein undilute was determined by dividing the corrected absorbance by 0.0542 and then multiplying that quotient by the dilution factor.

The absorbances, corrected absorbances, and calculated concentrations of the wildtype Asc Hb are as follows:

Sample Number Dilution Factor Absorbance Corrected Absorbance Calculated Concentration (μg/mL) Calculated Concentration (mg/mL)
1 20 2.049 1.431 528.044 0.528
2 40 1.575 0.957 706.273 0.706
3 100 1.088 0.47 867.1586 0.867
4 200 0.861 0.243 896.679 0.897
5 1000 0.638 0.02 369.004 0.369
6 2000 0.63 0.012 442.804 0.443

The two most reliable of these measurements are samples 3 and 4 because their absorbances fall within the range of absorbances taken for the standard today.

Spectra of Wildtype Asc Hb

  1. Spectra were taken from 200-800nm of the wildtype Asc Hb in a quartz cuvette. Spectra was taken of the protein concentrated, 2x dilute (500μL dH2O + 500μL concentrated protein), and 4x dilute (500μL dH2O + 500μL 2x dilute protein).
  2. The absorbances were corrected with the absorbances of water in the same quartz cuvette.
  3. The spectra were plotted using Excel:

Image:Wt_asc_hb_spectra_062712.JPG


The spectrum of just the 4x dilute protein was plotted:

Image:Wt_asc_hb_4x_dilute_spectrum_062712.JPG

Making Wildtype Asc Hb "Apo"

  1. 230μL of 1M HCl was added to 20mL of wildtype Asc Hb in 25mM Tris (pH 8).
    • This step hopefully lowered the pH of the protein to 2.3
  2. In the cold room 10mL of 2-butanone was added to the protein and the protein was shaken vigorously for 30 seconds.
  3. The solution then sat for about 5 minutes with no visible sign of separation. 10mL more of 2-butanone was added to the protein and it was shaken vigorously again for 30 seconds.
  4. The solution sat for about a minute and then a clear seperation was seen (the top half was pink and the bottom half was almost clear). The top half (the heme in 2-butanone) was removed by a Pasteur pipette.
  5. 20mL more of 2-butanone was added to the protein, shaken vigorously for 30 seconds, allowed to sit for a minute, and then when separation was seen the top half was removed by a Pasteur pipette.
  6. The previous step was repeated one more time.
  7. The protein layer was then transferred to dialysis tubing.
  8. The dialysis tubing was placed in 2L of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  9. The protein was dialyzed overnight (with stirring).


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