Help with trouble shooting: re-reading the same plate produces better results

[ORF1]

Hello, I'm doing Sanger sequencing and I'm getting some strange results that I've now reproduced at least 4 times. Every time I read my plate in the sequencer, my results look much better when I read it again the following day with absolutely no modifications to the plate. That is, multiple wells that were once negative and didn't show any signal, suddenly showed up as positive for my target on a re-read of the same plate on the next day. I'm using the BigDye xterminator kit with m13 primers after PCR. I'm also reading my plates on the 3730 DNA analyzer.

Does anyone know why this could be happening? I am at a complete loss as to what is going on.

Thanks in advance

[jrjong]

Hello, I'm doing Sanger sequencing and I'm getting some strange results that I've now reproduced at least 4 times. Every time I read my plate in the sequencer, my results look much better when I read it again the following day with absolutely no modifications to the plate. That is, multiple wells that were once negative and didn't show any signal, suddenly showed up as positive for my target on a re-read of the same plate on the next day. I'm using the BigDye xterminator kit with m13 primers after PCR. I'm also reading my plates on the 3730 DNA analyzer.

Does anyone know why this could be happening? I am at a complete loss as to what is going on.

Thanks in advance

Sorry for restarting an old thread, but i'm getting the same strange results. SO, if anyone knows the solution, it would be a great relief.

[Markiyan]

Basically use more 70% ethanol (clear, without fusel oils :-)

And spin at 10 degrees C (instead of 4).

See https://www.seqanswers.com/forum/seq...encing-results