User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/02

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Running a DNA gel to Extract and Purify PCR Clones

  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 10μL of DNA ladder was loaded into the first lane.
  3. 25μL of the wild-type PCR product with 5μL of 6x loading dye was loaded into the first wide lane.
  4. 25μL of the triple mutant PCR product with 5μL of 6x loading dye was loaded into the second wide lane.
  5. The gel was run at 90V 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 Wizard® SV Gel and PCR Clean-Up System Protocol

The full protocol can be found here.

  1. The gel was viewed a photographed under UV light.
    • Image:Photo_(24).JPG
  2. 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
  3. 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).
  4. 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
  5. 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.

  1. For the pQE-80-L-Kan, mix 27μL of DNA with 5μL of 10x NEBuffer2, 13μL of sterile dH2O, and 5μL of HindIII enzyme.
  2. For each PCR clone, mix 35μL of DNA with 5μL of 10x NEBuffer2, 5μL of sterile dH2O, and 5μL of HindIII enzyme.
  3. Incubate for two hours at 37°C.
  4. Incubate for 20 minutes at 65°C.
  5. Store at -20°C.

Purifying Reconstituted Asc Hb

Dr. Hartings continued to reconstitute Asc Hb with Cobalt over the weekend.

  1. 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
  2. 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.
  3. One column volume of dH2O was run over the column.
    • The flow rate of the column is gravity.
  4. Two column volumes of 25mM Tris, pH 8.3 was run over the column.
  5. The dialyzed Reconstituted Asc Hb was then run over the column.
  6. 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.
  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.


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