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Template Preparation for Automated DNA Sequencing..
Our lab routinely produces high quality data with read lengths to 700 bases; however, this depends on many factors, including how clean the template DNA is, or the annealing efficiency of the primer. To prepare DNA which is clean enough for automated sequencing, please take a careful look at this page!

Culture Conditions

Preparation methods

Preparation Kits recommended

Clean-up of DNA prepared by "old-fashioned" methods

Clean-up of PCR products

  1. Culture Conditions:

    • Do not overgrow E. coli cells. Overgrowth can result in release of chromosomal DNA and polysaccharides that co-purify with plasmids and inhibit sequencing reactions. We have been told that richer media, especially buffered ones, delay this process
    • A 0.5 M NaCl wash of the cells prior to lysis can be very helpful in removing polysaccharides and media components that often copurify with the DNA and inhibit sequencing reactions. Simply resuspend the cell pellet completely by vortexing, then re-spin.

  2. Preparation methods:

    • Plasmid templates can be prepared using a variety of protocols. We have found that purification columns designed specifically for Automated Sequencing work best (see below). However with a certain amount of trouble-shooting standard alkaline lysis, boiling, or CsCl methods are acceptable. The cleaner the DNA, the better the sequence!
      Miniprep kits do not ensure cycle-sequencing grade DNA!

  3. Preparation Kits recommended:

    • (NOTE: all QIAGEN/miniprep products are not equal)

    • QIAwell Plus/UltraPlasmid Kit (cat #16142, 16144)

    • QIAwell Plasmid purification system (cat#171220, 17124)

    • QIAGEN Plasmid Mini kits (cat# 12123 or 12125)

    • Promega Wizard Plus SV minipreps (cat#A1340)

    • ABI PRISM Miniprep kit (cat #402789)

  4. Clean-up of DNA prepared by "old-fashioned" methods:

    • For standard alkaline or boiling minipreps, it is important to avoid the co-precipitation of inhibitors from the cell lysate, especially when columns are not used for purification.
    • Use only the minimal amount of room temperature isopropanol (do not chill) for precipitation (0.60 volumes). Do not wait to spin and do not spin longer than 5 minutes to precipitate the DNA.
    • For many samples, including DNA precipitated from CsCl solutions (dialysis is better), one or more ammonium acetate precipitations are helpful to purify the DNA. Add 1/2 volume of 7.5 M ammonium acetate, mix well, then add 0.6 volumes of room temperature isopropanol, mix well, and spin. Residual CsCl in DNA samples will cause failures!
    • The majority of the RNA should be removed from miniprep DNA, by using RNAse, high salt precipitation or column purification.

  5. Clean-up of PCR products

MCF Home SDSU CSUPERB Contact Us Last Updated: 5/2006