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Bacteria assay for dna cleavage repair

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#1 Buddy



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Posted 05 December 2009 - 02:33 PM

I am trying to assay several proteins that can recognize and repair a specific break in a plasmid in ecoli. I am trying to design an assay that will allow me to cotransfect my repair protein plasmid and a target plasmid(with a double strand break) and would like to set the assay up so that I can monitor how effective the repair protein is by determining a ratio between repaired target plasmids and un-reparied target plasmids.

First of all, has anybody come across an assay that would answer such a question in vivo? Biochemical assays are a pain because my proteins are very hard to purify.

The main theoretical problem with this experimental design seems to be that the target plasmid(bearing dsDNA break) will be repaired by a bacterial ligase so efficently that I will not see a difference in number of repairs from the repair protein or the ligase.

I would appreciate your help.


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