Particle bombardment

Sharp Tool sharp.tool at bigpond.net.au
Wed Nov 16 17:41:11 EST 2005


  "Michael Sullivan" <mlsulliv at wisc.edu> wrote in message news:mailman.301.1132178285.29584.methods at net.bio.net...


  On Nov 16, 2005, at 2:24 AM, Sharp Tool wrote:


    Not sure how you are able to achieve 0.5ug/ul with QIAPreps.
    The handbook indicates alot less than that.

    Cheers
    Sharp Tool



  I guess it depends on what you mean by "a lot" less. The handbook I have indicates that you can expect 5-15 ug of DNA for a high copy plasmid like Bluescript from a 1.5 ml overnight culture. One can use more culture than that, and the columns have a binding capacity of 20 ug, so a concentration of 500 ng/ul (a yield of 25 ug) might be a tad high, but not so very much higher than indicated in the manual.


  Yield will of course depend a lot on the vector, the insert, the host, and growth conditions. Without knowing these things, it's hard to offer suggestions.


  Mike


  ---
  Michael L. Sullivan
  Plant Research Molecular Geneticist
  US Dairy Forage Research Center
  ARS-USDA
  1925 Linden Drive West
  Madison, WI 53706
  (608) 890-0046 (Phone)
  (608) 890-0076 (FAX)


  The original thread had a typo in it. The concentration I would like to achieve is 0.5 to 1.0 ug/ul.
  The concentration I'm getting is 0.05 ug/ul, which is consistent with the yield expected from the Qiagen handbook.

  Conditions:
  10ml LB overnight (16hrs) culture 38deg with shaking.
  5ml is used for miniprep.
  High copy plasmid in XL1-Blue.

  Sharp Tool
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