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Cre recombinase question

Cre recombinase question - Protocols and Methods Forum

Cre recombinase question - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-11-2008, 09:22 AM
Scott Brown
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Default Cre recombinase question



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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Old 07-11-2008, 07:50 PM
peter
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Default Cre recombinase question

On Jul 11, 5:22*am, "Scott Brown" <[Only registered users see links. ].edu.au> wrote:

Oh my, isnt is just easyer to sequence the plasmid, or PCR with cre
primers?
-Peter
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