Team:Nagahama/Experiments

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Protocols



Agarose gel

Agarose gel



  • Weigh 0,3 g of agarose.
  • Add 30 mL of TAE 1X.
  • Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
  • While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
  • When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
  • Pour the solution into the bed and clear all its bubbles with a tip. Put a piece of paper on top of it and let it polymerize.
  • Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.

    • </ol> Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.


    LB medium (1L liquid)


    • 10 g tryptone
    • 10 g NaCl
    • 5 g yeast extract
    • Water




    LB medium (solid, 1L = 50 dishes)



    • 15 g agar agar
    • 10 g tryptone
    • 10 g NaCl
    • 5 g yeast extract
    • Water

    For selective medium, suplement with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin ).





    Genome extraction



    For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to <a href="https://2014.igem.org/File:Easy-DNA_Kit.pdf" target="_blank">manufacturer's instructions</a>.




    Plasmids extraction



    For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to <a href="https://2014.igem.org/File:Miniprep.pdf" target="_blank">manufacturer's instructions</a>.





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