User:Floriane Briere/Notebook/CHEM-496/2011/09/27

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Objective

We are going to conduct two different experiments:

  • We are going to digest the mutated DNA we obtained thanks to the PCR reaction to isolate methyled DNA. Then, this DNA will be transferred into cells where it'll be expressed. If the experiment works, we should be able to observe fluorescent cells through the microscope (thanks to the GFP gene which is located in our DNA molecule).
  • We are going to synthetize Gold NPs again. According to our previous experiments, we observed the formation of purple filaments, this can be explain by the fact that the solution is taken out of the oven every time we make a spectrum. The solution doesn't stay at a stable temperature during the whole experiment, which can explain the low level of Gold Nps synthesis. During today's experiment, we are going to leave to solution at a stable temperature during the whole experiment.

Protocol

  • Gold NPs synthesis:
  1. Add 0.97ml of BSA (15.4µM) + 0.58ml HAuCl4 (4.3mM) + 8.45 buffer (50mM Tris;pH 7.5)
  2. We obtain a 10ml solution ([BSA] = 1.5µM ; [HAuCl4] = 0.25mM)
  3. Take a spectrum at T0
  4. Place the solution in the oven at 80°C during 2 hours
  5. Take spectrum every 30 minutes
  • DNA transformation into cells:
  1. Add 1µL of DpnI enzyme to the PCR solution (which containe the wild-type DNA)
  2. Make the LB/agar plate:
    1. The 200ml stock solution has been made by adding Agar (5g) and LB powder (5g); the agar/LB plate is made with LB powder which is full of nutriments; it will allow cells to live and grow
    2. Let the solution become solid
    3. Place the solution into the oven to make it become liquid again
    4. Put some solution into a plastic Petri dish
    5. Let the solution become solid
    6. Add antibiotic Ampicillin (100µg/mL)
  3. Prepare the cells:
    1. Place the DNA solution in the ince during 15 minutes
    2. Add 5µL of DNA solution to 40µL of cells solution
    3. Incubate on ice during 30 minutes
    4. Place the solution at 42°C for 30 seconds to make a heat shock ; the heat shock favors the transformation of the DNA into the cells
    5. Incubate in ice for 5 minutes
    6. Add 250μL of SOC media
    7. Incubate in shaker at 37°C for 1 hour
  4. Spread 100µL of cells on the LB/agar plate
  5. Store plate (inverted) at 37°C in a oven during 24 hours


Results: Gold NPs synthesis

  • Curve of the Absorbance as a function of the wavelength:

Image:27sept - Absorbance =f(wavelength).png

  • Curve of the Absorbance as a function of the wavelength (zoomed in the 550nm area):

Image:27sept - Absorbance =f(wavelength) - zoom.png

  • Curve of the Absorbance at 550nm as a function of time:

Image:27sept - Abs(550nm) = f(time).png

  • Conclusion:

At the end of the experiment, we obtained an incolore solution.

We can observe that there is no significant increase of the Absorbance at 550nm during the experiment. Compare to previous results, we can say that Gold NPs synthesis is not efficient in Tris buffer. This result is quite similar to the one we obtained when we used acetate as a buffer (on the 7th of september). We obtained better results when we used water as a buffer (on the 31st of august). The next experiment is to make the same protocol, using water as a buffer, but with a stable temperature.

Results: DNA's transfer into cells

The first step of the experiment was to add Dpnl enzyme to the PCR solution. This step allowed us to digest unmethylated DNA; we selected methylated DNA for the transfer.

Then, we transferred the DNA into novablue cells; cells which integrate our DNA molecules are then selected by adding Ampicillin to the agar/LB plate. In fact, the DNA molecule contains an antibiotics resistant gene.

The cells were then placed into a favorable environment to allow them to grow up.

After 24hours of incubation, we observed no cells on our plate.



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