IGEM:MIT/2005/Excising DNA from gels, purifying DNA from gels

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