Cloning advice - (May/21/2013 )

I clone a 915bp genes into PCDNA3. Because the RE I use is EcoR1 and XbaI, I change the stop codon of TGA into TAG due to dam methylation activity of XbaI. However, my sequencing result using T7 and BGH shows that my stop codon is lost. What could be the factor contributing it?

Thanks!

have you repeated the sequencing in both directions and verified the result?

Yeap, I have verified both ends results with T7 and BGH, still the same though..

sometimes you lose things during cloning. typically there is something unanticipated like a recombination event or poor primer design.

do you have other colonies you picked from the cloning to sequence?