plasmid-preparation

Daniel Jacobs d.jacobs at unsw.edu.au
Wed Jul 24 22:26:03 EST 1996


In article <R.B.Jansen.70.000DA56B at uni-bonn.de>, R.B.Jansen at uni-bonn.de
(Raymond B. Jansen) wrote:

> Hello!
> 
> How to increase the yield of a plasmid preparation of plasmids with a very 
> low-copy-RK2-origin (5 copies/cell)?
> I use the plasmid pBI 101 of the GUS-GENE-Fusion-System by CLONTECH 
> Company. I cloned my promotor (900bp) into the polylinker site of the pBI 101 
> (12.2 kb) and transferred it into JM 105 for amplification. The mini-plasmid 
> preparation with rapid-boiling-method or alkaly-lysis brought a yield of 100 
> ng DNA. So that was not enough for a restriction-analysis.
> How can I screen 400 cells without getting enough DNA?
> Please reply to: r.b.jansen at uni-bonn.de

Raymond try PCR your insert (900bp) directly from the colony (using
conserved primers at each end of the polylinker site) and then doing any
digest (if needed). If you pool your samples it shouldn't take more than
20 PCR reactions to find the right clone.



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