oliver.berkowitz at anu.edu.au
Wed Nov 16 19:10:13 EST 2005
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.
Dr. Oliver Berkowitz
The Australian National University
Research School of Biological Sciences
Environmental Biology Group
GPO Box 475
Canberra ACT 0200
P: +61 - (0)2 - 61254549
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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.
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.
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
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.
10ml LB overnight (16hrs) culture 38deg with shaking.
5ml is used for miniprep.
High copy plasmid in XL1-Blue.
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