Particle bombardment

[Oliver Berkowitz]

Hi, 

might also be overloading of the column. 5ml culture is quite a lot. But you'll never reach 1ug/ul, think a home made maxi prep might be the solution.

Cheers
Oliver

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Dr. Oliver Berkowitz 
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  "Sharp Tool" <sharp.tool at bigpond.net.au> wrote in message news:bAOef.19310$Hj2.14124 at news-server.bigpond.net.au...

    "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


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    Michael L. Sullivan
    Plant Research Molecular Geneticist
    US Dairy Forage Research Center
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    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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