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