User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/11

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Reconstitute Asc Hb with Manganese

The two dialysis tubes of apoprotein were combined for this reaction. The following procedure took place in a cold room (4°C):

  1. The apoprotein from the dialysis tubing was poured into a beaker and placed on a stir plate with a stir bar.
  2. 4mL of 1M Potassium Phosphate buffer (pH 7.1) was added dropwise (very slowly) to the protein (while gently stirring).
  3. Mn-porphyrin solution was added dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Mn-porphyrin solution was prepared by dissolving 2mg of Mn-porphyrin in 10 drops of 0.1N NaOH, and then adding 4.5mL of dH2O. 2mL of 0.1M Potassium Phosphate buffer (pH 7.1) was added to this solution. The solution was mixed on a stir plate.
  4. The beaker with the apoprotein/Mn-porphyrin mixture was wrapped in aluminum foil and left to gently stir in the cold room.

Running a DNA Gel

  1. A 1.2% agarose gel was made by mixing 0.3g of agarose with 25mL TAE buffer. This mixture was heated in the microwave for 37 seconds and then poured into a gel chamber with a comb inserted.
  2. When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
  3. 50μL of the PCR cloned wildtype Asc Hb from yesterday with 10μL of 6x loading dye was loaded into the first wide lane.
  4. A lane was skipped.
  5. 5μL of the wild-type Asc Hb/pQE-80-L-Kan ligation from last night with 1μL of 6x loading dye was loaded into the next lane.
  6. 5μL of the M8S/M33S/M103S Asc Hb/pQE-80-L-Kan ligation from last night with 1μL of 6x loading dye was loaded into the next lane.
  7. 50μL of the PCR cloned M8S/M33S/M103S Asc Hb from yesterday with 10μL of 6x loading dye was loaded into the second wide lane.
  8. The gel was run at 100V until the dye band had moved about 3/4 of the way down the gel.
  9. The gel was then stained in ethidium bromide for about an 30 minutes.

Image:Photo_(31).JPG

No evidence that the ligation did or did not work.

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 10μL of 1.46mg/mL BSA to 990μL of distilled water.
    • When more BSA was needed, this was repeated.
  1. Spectra from 500nm-700nm 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. 5μL of the concentrated protein was then mixed with 795μL of distilled water and 200μL of Bradford dye in a plastic cuvette.
  4. A spectrum of this mixture was taken from 500nm-700nm.
  5. The wildtype Asc Hb with Ni(protoporphyrin IX) protein at its unknown concentration was diluted by a factor of 10 by combining 5μL of the protein with 45μL distilled water. (this was the protein purified and concentrated on 07/09/12).
  6. 5μL of the 10x dilute protein was then mixed with 795μL of distilled water and 200μL of Bradford dye in a plastic cuvette.
  7. A spectrum of this mixture was taken from 500nm-700nm.
  8. 10μL of the 10x dilute protein was then mixed with 790μL of distilled water and 200μL of Bradford dye in a plastic cuvette.
  9. A spectrum of this mixture was taken from 500nm-700nm.
  10. 20μL of the 10x dilute protein was then mixed with 780μL of distilled water and 200μL of Bradford dye in a plastic cuvette.
  11. A spectrum of this mixture was taken from 500nm-700nm.

Bradford Results

BSA Standard Results and Curve

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 0.615.

Standard Concentration Absorbance Corrected Absorbance
Standard 1 0.73 μg/mL 0.7 0.085
Standard 2 1.46 μg/mL 0.747 0.132
Standard 3 3.65μg/mL 0.893 0.278
Standard 4 5.84μg/mL 1.021 0.406
Standard 5 8.03μg/mL 1.133 0.518
Standard 6 10.22μg/mL 1.221 0.606

Image:Bsa_standard_071112.JPG


  • The equation of this line was determined by Excel to be (forced through the origin) y=0.0639x, where y is the absorbance and x is the concentration.

Results for Wildtype Asc Hb with Ni(protoporphyrin IX)

  • 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 with Ni(protoporphyrin IX) itself would have an effect on the absorbance taken at 595nm, but it turns out the absorbance was negligible. The absorbance of 5μL of the concentrated wildtype Asc Hb mixed with 995μL dH2O in a plastic cuvette at 595nm was 0.056, and the corrected absorbance (subtracting the absorbance of 1mL of dH2O in a plastic cuvette at 595nm of 0.055) is 0.001. This is a very small effect, and the effect is negligible when the protein is diluted even more.
  • The concentration of the protein undilute was determined by dividing the corrected absorbance by 0.0639 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)
1 200 1.143 0.528 1652.582 1.65
2 2000 0.692 0.077 2410.016 2.41
3 1000 0.734 0.119 1862.285 1.86
4 500 0.883 0.268 2097.027 2.097
  • Sample 2's absorbance is out of the range of the absorbances of the BSA standard, so it might not be as reliable as the rest of the data.

Gel Purification of Wild-type and Triple Mutant Asc Hb Insert PCR Clones from Today's DNA Gel

The protocol for the Wizard® SV Gel and PCR Clean-Up System was followed: procedure. 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).

DNA ligation with T4 DNA Ligase

This will be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert. The procedure is based off of the protocol on the NEB website.

  1. Mix 2μL of 10x T4 DNA Ligase Buffer, 5μL of the double-digested pQE-80-L-Kan vector(from 07/04/12), 10μL of the double-digested insert (gel purified 07/10/12), 2μL 10mM ATP (prepared fresh today), and 1μL of T4 DNA Ligase.
  2. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.



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