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pcr amplify an insert in plasmid?


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#1 claritylight

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Posted 18 May 2011 - 02:57 AM

How does one pcr amplify an insert that integrated into the genome by site specific integration using a plasmid to transfect the cell?

#2 bob1

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Posted 18 May 2011 - 08:57 PM

Extract genomic DNA, do a PCR using specific primers...

If you need to look for the gene on the plasmid specifically, if for example the gene is also present in the cell natively, the anchor the primers in the plasmid sequence somewhere, or look for a plasmid specific gene such as Ampr or B-gal.

#3 Trof

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Posted 23 May 2011 - 02:56 AM

Question is if the insert integrated with a part of plasmid or not, for example with a loxP site. Claritylight needs to know the exact sequence that interated. If I recall correctly, usually insert contains selection marker (mamalian if transfecting mammal cells) to remove non-recombinated clones in culture. Maybe try to amplify that.

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