User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/10

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Continuing to Make Wildtype Asc Hb "Apo"

  1. Move the dialysis tubing from the overnight dialysis solution to a fresh solution of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  2. Dialyze for about four hours in this solution with stirring.
  3. Then move the dialysis tubing from the 10mM NaHCO3 dialysis solution into 2L of dH2O.
  4. Dialyze for about four hours in the dH2O with stirring.
  5. Move the dialysis tubing from the dH2O into 2L more of dH2O.
  6. Dialyze overnight in the dH2O with stirring.

The Wizard® SV Gel and PCR Clean-Up System

The procedure from Promega was followed starting where I left off yesterday for the wild-type PCR clone Asc Hb and triple mutant PCR clone Asc Hb. 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).

Cutting with BamHI for Cloning

  1. Mix 45μL of yesterday's HindIII digest products with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  3. Add 5μL of 10% SDS solution (for a final concentration of 0.91% SDS).
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.

Making Media

  • 250mL of SOB was made in a 2800mL flask by combining 5g of tryptone, 1.25g of yeast extract, 0.12g of NaCl, 625μL of 1M KCl, and 250mL of distilled H2O. Two of these were made and both were autoclaved on a liquid cycle. When cooled 1.25mL of 2M MgCl2 (autoclaved) was added to each flask.
  • Also autoclaved a dry cycle and then autoclaved some old plates today.

PCR for Cloning

The procedure is being repeated so that I will have more insert DNA available for double digests and ligations. This procedure was done for the wild-type Asc Hb (Hb pET 3d A) and the triple mutant Asc Hb (M8S/M33S/M103S):

  1. Mix 34.5μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL forward primer, 2.5μL reverse primer, and 1μL of either plasmid.
  2. Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
  3. Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
  4. Start with 30 seconds at 98°C on the thermocycler.
  5. Cycle through the following 40 times:
    • 10 seconds at 98°C
    • 30 seconds at 62°C
    • 30 seconds at 72°C
  6. A final extension step of 5 min was done at 72°C.
  7. The thermocycler was then held at 4°C.

Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.

Running a DNA Gel

  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 42 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 double digested cloned wildtype Asc Hb from this morning with 10μL of 6x loading dye was loaded into the first wide lane.
  4. A lane was skipped.
  5. 5μL of the wild-type PCR clone Asc Hb from 06/28/12 with 1μL of 6x loading dye was loaded into the next lane.
  6. 5μL of the M8S/M33S/M103S PCR clone Asc Hb from 06/28/12 with 1μL of 6x loading dye was loaded into the next lane.
  7. 50μL of the double digested M8S/M33S/M103S Asc Hb from this morning with 10μL of 6x loading dye was loaded into the second wide 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 an hour (the double digest bands are very light).

Image:Photo_(30).JPG

Starting Growth for Making Competent Cells

I will attempt to make competent cells again with both the BL21(DE3) and NovaBlue strains of E.coli. 3 scrapes (the cells were still frozen so I scraped the top of the frozen cells three times with three different sterile, wooden sticks) of each strain were added to 250mL of SOB. The flasks were placed at 18°C at 250rpm to grow.

Gel Purification of Double-Digested Wild-type and Triple Mutant Asc Hb Inserts 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, 7μL of the double-digested pQE-80-L-Kan vector(from 07/04/12), 10μL of the double-digested insert (gel purified 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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