Running an Analytical DNA Gel of HindIII Products from Yesterday
- 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.
- When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
- 5μL of HindIII cut pQE-80-L-Kan mixed with 1μL of 6x loading dye was loaded into the second lane
- 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).
- 5μL of the wild-type clone PCR product with 1μL of 6x loading dye was loaded into the fourth lane.
- 5μL of the M8S/M33S/M103S HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the fifth lane.
- 5μL of the M8S/M33S/M103S clone PCR product with 1μL of 6x loading dye was loaded into the sixth lane.
- The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
- The gel was then stained in ethidium bromide for about 30 minutes.
The DNA gel:
Close-up of wild-type and M8S/M33S/M103S Asc Hb bands:
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.
- 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.
- 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
- The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
- 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
- Mix 40μL of yesterday's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
- Incubate for two hours at 37°C.
- 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
- 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.
- When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
- 45μL of the double digested wildtype Asc Hb with 9μL of 6x loading dye was loaded into the first wide lane.
- 5μL of the wild-type HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
- 5μL of the wild-type clone PCR product with 1μL of 6x loading dye was loaded into the next lane.
- 5μL of the M8S/M33S/M103S HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
- 5μL of the M8S/M33S/M103S clone PCR product with 1μL of 6x loading dye was loaded into the next lane.
- 45μL of the double digested M8S/M33S/M103S Asc Hb with 9μL of 6x loading dye was loaded into the second wide lane.
- The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
- The gel was then stained in ethidium bromide for about 30 minutes.
The gel:
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
- 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.
- When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
- 45μL of the double digested pQE-80-L-Kan with 9μL of 6x loading dye was loaded into the first wide lane.
- 5μL of the midiprepped pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the next lane.
- The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
- The gel was then stained in ethidium bromide for about 30 minutes.
The gel:
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:
Making Wildtype Asc Hb "Apo"
This procedure was done twice, with two 20mL portions of the wildtype Asc Hb:
- 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
- In the cold room 20mL of 2-butanone was added to the protein and the protein was shaken vigorously for 30 seconds.
- 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.
- 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.
- The previous step was repeated one more time.
- The protein layer was then transferred to dialysis tubing.
- The dialysis tubing was placed in 2L of 10mM NaHCO3.
- Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
- 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.
- 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
- The large bands on the gel were cut out of the gel with a razor blade (additional safety required with UV light).
- 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
- The microcentrifuge tubes will be stored at 4°C overnight.
|