DNA Preps for Automated Sequencing

Michael W. Smith mws%molly at SDSC.EDU
Thu Apr 23 14:44:01 EST 1992


In reply to:
>Does anyone have a "fail-safe" method for the preparation of plasmid DNA from
>plasmids such as pGEM5zf(+) (Promega) for sequencing in an Automated >Sequencer.
>I have tried several methods ranging from CsCl DNA preps to Qiagen tips
>to alkaline lysate preps,  all with varying degrees of success.   However,  
>none of these methods seem to be "fail safe",  rather they appear to rely on
>some unknown factor!!  (maybe the position of the moon?).
>Anyway,  if someone does have a good method,  I would appreciate a 
>reply.
>	Cheers,
>
>	Gordon Munro
>	Dept. Cell. and Molecular Biology
>	University of Auckland
>	New Zealand
     We have been trying several different methods of prepping plasmid DNA for our ABI automated sequencer.  The easiest method has been the Promega "Magic" miniprep
which cost about one dollar each.  Genomic DNA and RNA contamination is usually
absent in the samples and most importantly they consistently sequence on our ABI.
We have also tested a AUTOGEN robot and obtain plasmid DNA which reliably sequences.
I have a feeling that many of the times that our reactions fail it is due to
overestimated plasmid DNA concentrations.  Perhaps this is the unknown factor 
related to the position of the moon.  

The brief answer is to try the Magic Minipreps.  I have isolated ~10ug of DNA from 
eighteen samples in 45 minutes once when I timed the process.  Hope that this 
information helps.

Good Luck,
mike
   *********************** Michael W. Smith **************************
  *             Salk Institute for Biological Studies               *
  * Molecular Genetics Laboratory     phone: (619)453-4100, ext 376 *
  * P. O. Box  85800                  fax:   (619)558-9513          *
  * San Diego, CA   92138             E-mail: mws at molly.sdsc.edu    *
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