IGEM:MIT/2005/Wednesday, June 8th

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