IGEM:MIT/2005/Wednesday, June 8th

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MIT iGEM 2005 Laboratory "Bootcamp" Day 3 (run by Natalie Kuldell and Kate Bacon Schneider)

I. Loading/running agarose gels

  • Background Information:
    • use of sample buffer (SB) with tracking dye (how far has it run?) and glycerol (to sink samples into wells of gel)
    • demonstration of how to remove comb and load gels
    • Run gel toward positive pole (because DNA has negatively charged phosphate backbone)
    • Molecular weight standards allow you to tell the size of fragments; some useful ones we used were:
      • 1 kb ladder for higher range (1-4 kb)
      • phiX174/HaeIII digest for lower range (200 bp-1.3 kb)
  • Exercise:
    • Students loaded their PCR reactions (Day 1), uncut plasmid DNA, digested plasmid DNA (Day 2), and MW standards on gel
    • ran gel @ 100V to about 1/2 way-->took photo

II. Excising DNA from gels

  • Background Information:
    • use long wave UV when excising bands from gel—less energy, less damage to DNA
    • protect eyes and face with face shield—NO SUNBURN!
    • change razor blades between each DNA fragment (prevents cross contamination)
  • Exercise:
    • students excised "vector" and "insert" bands from double digested pSB1A3 with help from staff

III. Purifying DNA from gel

  • Exercise:
    • students removed agarose/purified DNA using Qiagen gel extraction kit as specified in protocol
    • DNA was saved at 4˚C for use in ligations/transformations