plasmid integrated or not ?
smithwhi at students.msu.edu
Thu Mar 17 16:23:00 EST 1994
In Article <2m30lr$mi3 at agate.berkeley.edu> "Theo at mendel.Berkeley.EDU (AT)" says:
> The following question was posted to me by a friend. I told
> him to use inverse PCR (i.e. shearing the DNA into small fragments
> -> self ligation -> inverse PCR -> subcloning and sequencing
> -> designing new primers -> PCR across the junction betwwen
> the vector and genomic sequence using unsheared genomic DNA
Why do the second PCR step? If the product of the first PCR is larger
than the distance between the two primers, what other interpretation could
you generate? Would the second PCR be able to discriminate between this
interpretation and integration in the genome?
> as templates ). Does anyone on the net have better
> idea(s) ?
> How can you test if the plasmid vector is integrated into
> the genome of transfected cells ? (Only 5% of cells are
> transfected and there's no selection marker on the vector).
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