Hello everyone!

I am thinking to amplify T-DNA insertion region using LBb1.3 (left T-DNA border) and RP (gene specific Reverse Primer) against the genomic DNA of one of the T-DNA insertion mutant line.

According to T-DNA insertion coordinates (which is roughly estimated to span between 300-400 bp in coding region of my gene) the RP is gene specific primer designed by me unlike RP (Right Genomic Primer) suggested by SALK. The RP which I want to use is downstream to T-DNA insertion and thus I expect an amplicon which would have T-DNA sequence along with my gene fragment. For information, the RP I am going to use is ~500bp downstream to edge of T-DNA insertion.

As I have discussed with my colleagues I was told that amplification would result in very long fragment and Its not feasible to get a PCR product.

I want to know the expert opinion in case if someone has done it before?

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