Is it possible to use inverse pcr? ie cut the inserted sample genome with a common cutter that does not exist within the insert. religate under self annealing (low concentration) conditions and then amplify the unknown sequence with primers internal to the insert but facing outwards from the insert ( therefore inwards to the circularised sequence thus generating the flanking sequence. Alternatively NGS

This seems like a very attractive solution. I will look more into it.

Thank you!

...in addition to the idea supposed by Paul Rutland you may think about:

1. a similar restriction digest but the ligation of a sequence not present in the mouse genome on the overhangs and a PCR using a forward primer in your inserted gene and a reverse primer targeting the ligated sequence.

2. depending on the inserted gene structure and resulting transcripts you may use a 3´ RACE RT-PCR. Similar to the first point you add a specific sequence to the poly-dT primer and amplify with a forward primer of your inserted gene and reverse primer targeting the 3´-end of transcripts. Potentially you may get a PCR product/sequence of a fusion transcript of your gene and neighboring sequences from the mouse genome.

Best,

Norman