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Problem with Sanger sequencing: re-reading of the plate produces better results?

ORF1
in Biochemistry and Molecular Biology

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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.


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


Thanks in advance


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