User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/28

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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.

PCR for Cloning

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
    • 15 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 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 an hour.

Beginning the 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.
  2. Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
    • Tube for wild-type PCR: 1.05g
    • Tube for triple mutant PCR: 1.03g
  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: 1.38g
    • Tube for triple mutant PCR: 1.25g
  5. The microcentrifuge tubes will be stored at 4°C overnight.

Transformations

Transformations are going to be done with the uncut pQE-80-L-Kan and double digested pQE-80-L-Kan, to make sure the pQE-80-L-Kan was cut previously on 06/15/12. The uncut plasmid (the midiprep product from 06/19/12 should transform, and the double-digested plasmid should not if it's actually cut.

  1. Place plastic culture tubes on ice for about 15 min.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 1uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (30uL, 40uL, or 50uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour.
  10. Plate 50uL of culture media on LB/amp plates (prepared 6/27/12: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, left in the autoclave overnight, so to pour plates today I had to melt the LB/agar over a Bunsen burner flame, then I added 250μL of 100mg/mL ampicillin).
  11. Incubate inverted overnight at 37°C.

Overnight Cultures

  • For mini-prep: scrape NovaBlue + M8S/M33S/M103S glycerol stock with sterile wooden stick and inoculate 10mL of LB with 100μg/mL ampicillin, grow overnight at 37°C at 250rpm
  • For making competent cells: scrape NovaBlue frozen competent cells (from -80°C freezer) with sterile wooden stick and inoculate 10mL of LB, grow overnight at 37°C at 250rpm


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