Team:GO Paris-Saclay/protocols


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PROTOCOLS

  • Dissolve the LB-agar in the microwave
  • Cool the agar down to room temperature until you can hold the bottle by hand.
  • Add the correct amount of the correct antibiotic.
  • Under sterile conditions, pour the LB-agar in empty Petri dishes.
  • Leave the Petri dishes open until the agar has solidified
  • Prepare the mix for all the samples in a single 1.5 mL tube. For one sample

    • tapro1

  • Add the total volume per sample (without DNA) to each PCR tube.
  • Add the template DNA to each tube.
  • Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

    • tapro1

  • Run a DNA electrophoresis following the DNA electrophoresis protocol.

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  • Decide on which enzyme(s) to cut with. Check online what buffer the enzyme(s) work(s) in. For Bio labs enzyme use the Double Digest Finder.
  • Prepare a sample as follows:

    • tapro1

  • Incubate for one hour at 37 °C.
  • Turn on the NanoDrop
  • Press the button for dsDNA to measure the concentration of double-stranded DNA in your samples.
  • Clean the measurement surface with a piece of tissue and ethanol.
  • Use the elution buffer or the media where your DNA is contained as a blank.
  • Clean the measurement surface with a piece of tissue and water.
  • Use 2 µL of sample to measure its concentration.
  • If you have multiple samples, clean the measurement surface in between measurements.
  • Pour the solution Agarose (0, 8%)-EtBr into the mould. Add a comb to create wells for the samples. Let it solidify (approx. 20 minutes).
  • Transfer the gel to the electrophoresis Remove the combs and cover it with TAE (0, 5 X). Transfer the gel to the electrophoresis.
  • Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5 µL works fine) and load 5-10 µL of the samples in the other wells.
  • Use the elution buffer or the media where your DNA is contained as a blank.
  • Run at 100 V for 25 min
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