Cre recombinase question

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Fri Jul 11 14:50:01 EST 2008


On Jul 11, 5:22 am, "Scott Brown" <SBr... from ccia.unsw.edu.au> wrote:
> Hi,
>
> I have performed a few digests on what I am pretty certain is a Cre plasmid however none of the fragments match up to the plasmid map.
>
> To validate that i do infact have Cre, I was thinking of doing a cotransfection, transfecting one population of 293's with pCre and another with a trap construct that contains 2 loxP sites I want to utilise in the future.
>
> If i combine the 2 populations of cells, will the cre being produced act on the loxP sites in the other population of cells to drop out the cassette flanked by each?
>
> If that is the case how long would this take? once i was certain the cre had been given enough time to act would i then put the cells under selection pressure to ensure a pure population of cells containing the now modified trap construct?
>
> Then extract the rna, run a pcr using primers designed for the 5' and 3' ends of the segment containing the loxP sites, run this on a gel, if i get no bands will i be able to conclude the cre works, provided i include a positive control that produces fragments using these primers?
>
> If anyone has any suggestions or modifications to this protocol i'd be very appreciative.
>
> Kind Regards
>
> Scott Brown
> PhD Candidate
> Children's Cancer Institute of Australia
>
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Oh my, isnt is just easyer to sequence the plasmid, or PCR with cre
primers?
-Peter


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