User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/03

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Running an Analytical DNA Gel of HindIII Products from Yesterday

  1. A 2% agarose gel was made by mixing 0.5g 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. 5μL of HindIII cut pQE-80-L-Kan mixed with 1μL of 6x loading dye was loaded into the second lane
  4. 5μL of the wild-type HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the third lane (the lane after skipping the wide lane).
  5. 5μL of the wild-type clone PCR product with 1μL of 6x loading dye was loaded into the fourth lane.
  6. 5μL of the M8S/M33S/M103S HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the fifth lane.
  7. 5μL of the M8S/M33S/M103S clone PCR product with 1μL of 6x loading dye was loaded into the sixth lane.
  8. The gel was run at 200V 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 30 minutes.

The DNA gel: Image:Photo_(22).JPG


Close-up of wild-type and M8S/M33S/M103S Asc Hb bands: Image:Photo_(23).JPG

The wild-type HindIII cut Asc Hb moved into the next lane a bit and is much lighter, but it still looks as though both of the HindII cut clones are shorter than their original PCR products.

Continuing Purification of Reconstituted Asc Hb

I will continue the purification of the Reconstituted Asc Hb that was started yesterday. I will start with a new column again today.

  1. A PD-10 Desalting column was prepared with one column volume of dH2O was run over the column, and two column volumes of 25mM Tris, pH 8.3 run over the column.
  2. 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
  3. The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
  4. The loading and elution of the protein was repeated many times.
  • This procedure was repeated with a new column when the first column started to appear too dirty (the excess Co(protoporphyrin IX) did not elute off the column).

Cutting with BamHI for Cloning

  1. Mix 40μL of yesterday's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  3. Add 4μL of 10% SDS solution (for a final concentration of 0.89% SDS).
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.

Running an Analytical/Gel Purification DNA Gel of Asc Hb HindIII/BamHI Products from This Morning

  1. A 2% agarose gel was made by mixing 0.5g 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. 45μL of the double digested wildtype Asc Hb with 9μL of 6x loading dye was loaded into the first wide lane.
  4. 5μL of the wild-type HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
  5. 5μL of the wild-type clone PCR product with 1μL of 6x loading dye was loaded into the next lane.
  6. 5μL of the M8S/M33S/M103S HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
  7. 5μL of the M8S/M33S/M103S clone PCR product with 1μL of 6x loading dye was loaded into the next lane.
  8. 45μL of the double digested M8S/M33S/M103S Asc Hb with 9μL of 6x loading dye was loaded into the second wide lane.
  9. The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
  10. The gel was then stained in ethidium bromide for about 30 minutes.

The gel: Image:Photo_(25).JPG

There isn't a distinct difference between the single cut and double digested Asc Hb, but hopefully the second cut occurred.

Running an Analytical/Gel Purification DNA Gel of the pQE-80-L-Kan HindIII/BamHI Product from This Morning

  1. A 2% agarose gel was made by mixing 0.5g 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. 45μL of the double digested pQE-80-L-Kan with 9μL of 6x loading dye was loaded into the first wide lane.
  4. 5μL of the midiprepped pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the next lane.
  5. The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
  6. The gel was then stained in ethidium bromide for about 30 minutes.

The gel: Image:Photo_(26).JPG

It doesn't look like the cut occurred in the correct place (bright band looks like it has too few kb). This is similar to the gel seen on 6/25/12.

Spectra of Reconstituted Asc Hb with Co(protoporphyrin IX)

A spectrum was taken with 3mL of the protein collected from the purification this morning. Another spectrum was taken of this 2x dilute (1.5mL protein + 1.5mL 25mM Tris, pH 8.3). A spectrum was also taken of the blank, 25mM Tris, pH 8.3, and the absorbance values for the blank were subtracted from the absorbance values of the protein:

Image:Reconstituted_asc_hb_co_spectra_070312.JPG

Making Wildtype Asc Hb "Apo"

This procedure was done twice, with two 20mL portions of the wildtype Asc Hb:

  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 20mL of 2-butanone was added to the protein and the protein was shaken vigorously for 30 seconds.
  3. 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.
  4. 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.
  5. The previous step was repeated one more time.
  6. The protein layer was then transferred to dialysis tubing.
  7. The dialysis tubing was placed in 2L of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  8. The protein was dialyzed overnight (with stirring).

Beginning the The Wizard® SV Gel and PCR Clean-Up System Protocol

The full protocol can be found here.

  1. Three 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
    • Tube for pQE-80-L-Kan double digest: 1.03g
    • Tube for wild-type double digest: 1.03g
    • Tube for triple mutant double digest: 1.03g
  2. The large bands on the gel were cut out of the gel with a razor blade (additional safety required with UV light).
  3. The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
    • Tube for pQE-80-L-Kan double digest: 1.16g
    • Tube for wild-type double digest: 1.19g
    • Tube for triple mutant double digest: 1.21g
  4. The microcentrifuge tubes will be stored at 4°C overnight.



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