Running a DNA gel to Extract and Purify PCR Clones
- 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.
- When the gel had hardened the comb was removed and 10μL of DNA ladder was loaded into the first lane.
- 25μL of the wild-type PCR product with 5μL of 6x loading dye was loaded into the first wide lane.
- 25μL of the triple mutant PCR product with 5μL of 6x loading dye was loaded into the second wide lane.
- The gel was run at 90V 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 Wizard® SV Gel and PCR Clean-Up System Protocol
The full protocol can be found here.
- The gel was viewed a photographed under UV light.
- Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
- Tube for wild-type PCR: 1.03g
- Tube for triple mutant PCR: 1.04g
- The large bands on the gel (at ~0.5kb) were cut out of the gel with a new razor blade (additional safety required with UV light).
- The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
- Tube for wild-type PCR: 1.24g
- Tube for triple mutant PCR: 1.21g
- The procedure was then completed 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).
Cutting with HindIII for Cloning
This was done with both the wildtype Asc Hb and the M8S/M33S/M103S Asc Hb clones from the Gel Clean-Up earlier today and the pQE-80-L-Kan midiprep from 06/19/12.
- For the pQE-80-L-Kan, mix 27μL of DNA with 5μL of 10x NEBuffer2, 13μL of sterile dH2O, and 5μL of HindIII enzyme.
- For each PCR clone, mix 35μL of DNA with 5μL of 10x NEBuffer2, 5μL of sterile dH2O, and 5μL of HindIII enzyme.
- Incubate for two hours at 37°C.
- Incubate for 20 minutes at 65°C.
- Store at -20°C.
Purifying Reconstituted Asc Hb
Dr. Hartings continued to reconstitute Asc Hb with Cobalt over the weekend.
- Buffers were prepared:
- 25mM Tris, pH 8.3 was prepared with 3.03g Tris, pH with HCl, final volume is 1L with dH2O
- 25mM Tris, 1M NaCl, pH 8.3 was prepared with 3.03g Tris, 58.44g NaCl, HCL to pH, final volume is 1L with dH2O
- A CM Sepharose Fast Flow column was packed by resuspending the column in the solution it was in (20% ethanol), and then pouring it into the glass column. The column was allowed to settle and pack for a few hours.
- One column volume of dH2O was run over the column.
- The flow rate of the column is gravity.
- Two column volumes of 25mM Tris, pH 8.3 was run over the column.
- The dialyzed Reconstituted Asc Hb was then run over the column.
- Any colored liquid that came off the column was collected and saved. More 25mM Tris was poured onto the column until the flowthrough looked clear.
- Unfortunately it seems everything came off the column so another column will be used.
- 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.