User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/29

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Miniprep of M8S/M33S/M103S

A miniprep of M8S/M33S/M103S Asc Hb from the NovaBlue cells grown overnight was done this morning. The vacuum protocol was followed: [1]

The Wizard® SV Gel and PCR Clean-Up System

The procedure from Promega was followed starting where I left off yesterday. 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).

Making NovaBlue Cells Competent

  • The following procedure will be used to make competent cells: [[2]]. The changes to the procedure will be that HEPES will be used instead of MOPS (the buffer will still be made to the same pH) and the culture will be grown at 165rpm instead of 250rpm (because there are large flasks in the incubator as well).
  1. 1mL of the overnight culture was transferred to the pre-warmed flask and was incubated at 37°C at 165rpm.
  2. The mixture was grown for two hours.
  • The OD600 at this point was 0.7 (the OD600 was only supposed to go to 0.5).
  1. The mixture was placed on ice for 5 minutes and then transferred to 50mL centrifuge tubes (divided evenly between two tubes).
  2. The tubes were spun at 4000g at 4°C for 5 minutes. The supernatent was discarded.
  3. The pellets were resuspended in a total volume of 30mL of the TFB1.
    • 100 mM RbCl, 50 mM MnCl2, 30 mM Potassium acetate, 10 mM CaCl2, 15% glycerol, pH 5.8
  4. The resuspended pellets in buffer were then placed on ice for 90 minutes.
  5. The cells were spun at 4000g at 4°C for 5 minutes.
  6. The supernatent was discarded and the pellet was resuspended in 4mL of TFB2.
    • 10 mM HEPES, 10 mM RbCl, 75 mM CaCl2, 15% glycerol, pH 6.8
  7. The resuspended cells were alliquoted in 200μL amounts (with three 500μL amounts).
  8. The cells were placed at -80°C.

Reconstitute Asc Hb with Cobalt

The following procedure took place in a cold room (4°C):

  1. The apoprotein from the dialysis tubing was poured into a 50mL beaker and placed on a stir plate with a stir bar.
  2. 2mL of 1M Potassium Phosphate buffer (pH 7.1) was added dropwise (very slowly) to the protein (while gently stirring) over a 20 minute period.
  3. Co(protoporphyrin IX)solution was added dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Co(protoporphyrin IX)solution was prepared by dissolving 2mg of Co(protoporphyrin IX) in 10 drops of 0.1N NaOH, and then adding 4.5mL of dH2O. The solution was mixed by pipetting up and down.
  4. The beaker with the apoprotein/Co(protoporphyrin IX) mixture was wrapped in aluminum foil and left to gently stir in the cold room overnight.

Note: Dr. Hartings will continue to reconstitute Asc Hb with Cobalt over the weekend.

PCR for Cloning

The procedure is being repeated again today with a longer extension step in each cycle, so it will be easier to see the cut in the DNA to know the digests are working. 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.

Quantifying DNA

The absorbance of the midiprepped pQE80-L-Kan 06/19/12 was taken at 260 ans 280nm in order to calculate the DNA concentration.

  1. Using a smaller volume quartz cuvette, 80μL of dH2O was placed in the spectrophotometer and the baseline was corrected with this.
  2. 20μL of DNA was added to the 80μL of water. This was pipetted up and down with a pipette to mix.
  3. The absorbance was taken at 260 and 280nm.

Results

Note: the absorbances are for a 5x dilute DNA sample

double-digested pQE80-L-Kan

  • A260 = 0.148
  • A280 = 0.091
  • Absorbance Ratio = 1.6175
  • 20x Dilute DNA concentration = 7.4000 μg/mL
  • Non-dilute DNA concentration = 37 μg/mL


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