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How to determine the location of integrated DNA in stable cell lines

transfection stable transfection mapping human cells

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#1 kaveh

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Posted 19 September 2013 - 02:39 PM

When you make stably transfected cell lines, I assume that the plasmid DNA (or at least part of it) gets randomly integrated into the genome of the cells. How one can determine the exact location of the plasmid DNA in the host genome?

 

Thank you. 



#2 bob1

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Posted 19 September 2013 - 03:27 PM

The quickest way would be to anchor some primers in the plasmid and sequence out off the genomic DNA.  Otherwise you could create restriction fragments and clone libraries etc.



#3 phage434

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Posted 19 September 2013 - 05:19 PM

Inverse PCR. Cut the genomic DNA with a frequent cutter (4 bp or so). Dilute the sample, religate. This forms circular fragments cut at the frequent cutter site. Now PCR using outward directed fragments on your insert. It will amplify the flanking regions of the genomic DNA outside of your insert site. Sequence.







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