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METHODS OF DNA PURIFICATION The mere use of the kits we recommend does not guarantee success. You must still use good technique! 1. Qiagen Spin Kits (We highly recommend these kits. The gravity columns also work well but are more expensive.) In addition, Qiagen publishes a very good template purification guide. Please ignore its advice on genomic preparation as we do not accept genomic preps. If you are having trouble getting yield with the Spin kits: Elute with hot dH2O (60C-70C) that has been recently autoclaved. Also, do the optional wash even if your strain is not part of the JM series. 2. Promega Wizard SV . (Can be purchased through Fisher.) Although we don't know much about this kit, we highly recommend it as well. Those who have used it have gotten excellent results with it. 3. Genemate Mini Prep - Also tested by customers with reliable bench skills. Seems to work just as well as the Qiagen and is cheaper! 4. CsCl ISOLATION HAS BEEN MOVED TO THE NOT RECOMMENDED LIST! 5. Mini-alkaline lysis + PEG precipitation Note: This is labor intensive and requires some skill. If you don't have experience isolating plasmids, you might want to consider an alternative method or ask someone for help. 6. M13 single-stranded rescue (Most manual sequencing protocols will work.) If you choose this method, you will only need half the amount of DNA/rxn. (.5µg) 7. Bio-Rad Plasmid Miniprep Kit : 8. Nucleobond AX Cartridge Note : We have never tried this but it is endorsed by Applied Biosystems. 9. Novagen UltraMobius 200 Plasmid Kit : Our thanks to John Beard in Timothy O'Brien's lab for testing it! It works great! *********** Purification Methods NOT Recommended 1. Any Qiagen MIDI or MAXI kit! Some people can make these work for sequencing but we do NOT recommend them. They have proven to be unreliable and inconsistant for sequencing. NOTE: EtOH contamination is usually the problem with the MIDI kit. If you insist on using it, dry your samples down in a vacuum dryer and then resuspend them in the original volume. 2. S & S DNA Elution Beads 3. Stratagene Strataresin 4. Isoquick Plasmid Kit 5. Boiling 6. Classical Alkaline Lysis 7. 3'-5' Plasmid Isolation Kit. Although Perkin Elmer recommends it, it seems to be very inconsistant. 8. Gibco's Concert Prep. Although Gibco claims this will work, many customers who have changed to it started having problems. These customers were people with reliable bench skills. 9. BIO 101 Genekleen 10. CsCl purification Note: We used to recommend this for the 377 but it is not suitable for capillary sequencing.
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