Hi jfrankl1, Are you still having an issue with the Sanger-Sequencing of the repeated symmetric region? If so, what are your cycling parameters? Have you only done direct sequencing of the PCR product, or have you also put it in a vector to sequence it using the vector priming sites? If you would like to provide further details, you can email me at <email obscured>. Scott Herke Manager, LSU Genomics Facility
________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of jfrankl1 <email obscured>> Sent: Tuesday, November 30, 2021 6:28 AM To: <email obscured> <email obscured>> Subject: [ABRF Discussion Forum] Sanger Sequencing odd data...... β 1 file link β We are having a technical issue sequencing a repeated symmetric region. This PCR product is 332bp long and it includes three symmetric lacOs without gaps in between. The sequencing results suggest that when it finished the first lacO, it somehow "hopped" to the complement strand and sequenced back to the origin. We see this with both forward and reverse primer sequencing showing this "hopping" effect. Please see our attached figure for more information. We simply cannot figure out what is going on here. The data was verified by resequencing with a different facility. Please let us know if you have also experienced this before and have some solution to resolve issue! See attached simple drawing for example.... Thanks! β 1 file β β‘ InkedRepeatedSymmetricRegion_LI.jpg (2mb) https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ffile%2FtvHsCMUfuohOf679NBwGv3QSxwH-76Ex-2SP9A9f%2F&data=04%7C01%7Csherke%40lsu.edu%7C778e69016bd44587b1b808d9b3fce44d%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637738721004999221%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=ztRdKthLeQLYT4aOI%2FSH6PRtBmEEef%2Fju%2FkrJAcIEzY%3D&reserved=0 ββ View topic https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F3NPa1iQK6uevuiTIM0Q5pV&data=04%7C01%7Csherke%40lsu.edu%7C778e69016bd44587b1b808d9b3fce44d%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637738721004999221%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=B7Bbni2YP8Hotej5qL0J481KMheDr1YcEdqlW9VROIM%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe