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

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# We obtain a 10ml solution ([BSA] = 1.5µM ; [HAuCl4] = 0.25mM)
# We obtain a 10ml solution ([BSA] = 1.5µM ; [HAuCl4] = 0.25mM)
# Take a spectrum at T0  
# Take a spectrum at T0  
# Place the solution in the oven at 80°C
# Place the solution in the oven at 80°C during 2 hours
# Take spectrum every 30 minutes
# Take spectrum every 30 minutes


Revision as of 09:30, 28 September 2011

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Objective

  • Digest unmethyled DNA to isolate methyled DNA.
  • Gold NPs synthesis by using Tris buffer. During the experiment, the solution is left at 80°C during the entire 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)
    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
    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

Notes

  • We introduce the Ampicillin antibiotics into the agar/LB plate because our GFP gene is also composed of a resistant gene which allow the owner to resist Ampicillin. This step is going to allow us to select the cells which will have integrate the DNA fragment.
  • Type of cells : novablue cells
  • Vector : PRSET/DNGFP
  • Mutation: D1CGFP
  • The agar/LB plate is made with LB powder which is full of nutriments; it will allow cells to live and grow
  • The heat shock favors the transformation of the DNA into the cells

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.



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